Water-soluble fluoro-substituted cyanine dyes as reactive fluorescence labelling reagents

ABSTRACT

Disclosed are cyanine dyes that are useful for labelling and detecting biological and other materials. The dyes are of formula (I): 
     
       
         
         
             
             
         
       
     
     in which at least one of groups R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10  R 11 , R 12 , R 13  and R 14  is -L-M or -L-P, where L is a linking group, M is a target bonding group and P is a conjugated component, and at least one of groups R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9  and R 19  comprises fluorine. The use of cyanine dyes substituted by fluorine and having additional substitution with three or more sulphonic acid groups for labelling biological target molecules results in a labelled product in which there is reduced dye-dye aggregation and improved photostability, compared with cyanine dyes having no such substitutions. The dyes of the present invention are particularly useful in assays involving fluorescence detection where continual or repeated excitation is a requirement, for example in kinetic studies, or in microarray analyses where microarray slides may need to be reanalysed over a period of days.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.11/379,596 filed Apr. 21, 2006, which claims priority to patentapplication number 0508082.5 filed in Great Britain on Apr. 22, 2005 andto patent application number 0517656.5 filed in Great Britain on Aug.31, 2005; the disclosures of which are incorporated herein by referencein their entireties.

FIELD OF THE INVENTION

The present invention relates to the field of fluorescence labellingreagents, in particular, reactive cyanine dyes containingfluoro-substituents and to methods utilising such dyes.

BACKGROUND OF THE INVENTION

Cyanine dyes are widely used as reagents for fluorescence labelling ofbiologically important molecules such as proteins, nucleic acids,hormones and drugs. Indeed, cyanine dyes offer a number of advantagesover other fluorescent dyes. For example, the excitation and emissionspectra of cyanine dyes span the visible and near-infrared spectrum from450 nm to 800 nm. Furthermore, the cyanine dyes are characterised byhaving very high extinction coefficients and favourable quantum yields.See for example, U.S. Pat. Nos. 6,048,982, 5,268,486, 5,569,587,(Waggoner, A. S. et al). However, with certain cyanine dye structuresthere is a tendency towards self-association (or aggregation) leading tofluorescence quenching and a notable hypsochromic wavelength shift inabsorbance.

Recently, Waggoner et al (Org. Letters, (2004), 6(6), 909-912) hasdescribed a polyfluoro-thiadicarbocyanine dye (i) having goodphotostability in aqueous solvents. The dye exhibited reducedaggregation, enhanced quantum yield and greater resistance tophotobleaching when compared with a non-fluorinated analogue.

Modification of the indolinium ring of a carbocyanine dye at least oneof the 3-positions, so as to introduce a reactive group or a conjugatedsubstance has been described in WO 02/26891 (Molecular Probes Inc.). Themodified dyes according to WO 02/26891 have also been reported toovercome the tendency of cyanine dyes to self-associate and dyeconjugates labelled with the modified dyes are reported to be morefluorescent than conjugates labelled with structurally similarcarbocyanine dyes.

Japanese Patent Application No. 5313304 (Fuji Photo Film Co. Ltd.)discloses a silver halide photographic sensitive material incorporatinga dye containing multiple sulphonate groups and represented by formula(ii):

in which R¹⁰ and R¹¹ may be alkyl carboxylate or alkyl sulphonatemoieties and A includes:

Japanese Patent No. 61248789 (Ricoh KK) discloses an optical informationrecording medium including a recording layer containing a compoundhaving the formula (iii):

in which A is represented by the formula:

Y is a halogen atom; a is an integer from 1 to 4; R¹ and R² are a C₁-C₅alkyl group; R³ is a C₁-C₁₀ alkyl group which may be substituted by asulphonyl group or a carboxyl group; R⁴ is hydrogen, halogen, amino oralkanoyloxy, Z is an atomic group necessary for the formation of a 4-8membered ring, and m and n are integers from 0-3, where m+n≦3. Mader, O.et al (Bioconjugate Chem., (2004), 15, 70-78) describe the synthesis andphotophysical properties of a series of pentamethine indocyanine dyessubstituted at the aromatic ring positions with sulphonate, fluorine andmethyl.

None of the above documents discloses reactive cyanine dyes and cyaninedyes conjugated to a substance, wherein the dyes contain at least oneand preferably multiple fluoro substituents, as well as one or moresulphonic acid groups attached to the indolinium 1- and/or 3-positionsof the cyanine chromophore.

BRIEF DESCRIPTION OF THE INVENTION

The present invention provides cyanine dyes and components conjugatedwith such dyes, in which the cyanine dyes have the properties ofincreased photostability and reduced dye-dye interactions. The dyes asdescribed and claimed herein may be provided with at least one groupsuitable for direct covalent labelling of a target material. The dyes ofthe present invention are therefore particularly useful in assaysinvolving fluorescence detection where continual excitation is arequirement, for example in kinetic studies, or in microarray analyseswhere microarray slides may need to be reanalysed over a period of days.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 and FIG. 2 summarise the fluorinated dye compounds that have beensynthesized (Compound 1-Compound 21) with reference to Formula (II) andFormula (III);

FIG. 3 is a plot illustrating photostability data for Cy5F (Compound 1)and Compound 5. For comparison purposes, photostability data for thenon-fluorinated analogue Cy™ 5 is also presented (Cy™ is a trademark ofGE Healthcare Limited);

FIG. 4 is a plot showing photostability data for Cy7F (Compound 2),Compound 18 and Compound 19 compared with the non-fluorinated analogue(Cy7);

FIG. 5 is a plot showing photostability data for Compound 3 and Compound22 compared with Alexa® 647 (Invitrogen);

FIG. 6 is a plot showing photostability data for Compounds 5, 7, 9, 11and 13 compared with Alexa® 647 (Invitrogen) and Cy5;

FIG. 7 is a plot showing photostability data for Compounds 6, 8, 10, 12and 14 compared with Cy5;

FIG. 8 is a plot showing photostability data for Compounds 15, 16 and 17compared with Alexa® 647 (Invitrogen) and Cy5;

FIG. 9 is a plot showing photostability data for Compound 20 andCompound 21 compared with Cy3; and

FIG. 10 is a plot showing Compounds 1, 3 and 5-17 sorted in order ofphotostability for the T=96 hour data point.

DETAILED DESCRIPTION OF THE INVENTION

In a first aspect there is provided a compound of formula (I):

wherein:the groups R¹, R², R¹¹, R¹², R¹³ and R¹⁴ are selected independently from-L-M, -L-P, C₁-C₆ alkyl and —(CH₂)_(k)—SO₃H, where k is an integer from1 to 10; the groups R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹ and R¹⁰ are selectedindependently from hydrogen, -L-M, -L-P, —SO₃H, —SO₂—CF₃ and—(CF₂)_(m)—F, where m is 0 or an integer from 1 to 4; or R³ taken incombination with R⁴ or R⁵ taken in combination with R⁶ and/or R⁷ takenin combination with R⁸ or R⁹ taken in combination with R¹⁰ form a fusedaromatic six-membered ring containing carbon atoms and being optionallysubstituted one or more times by —SO₃H, —SO₂—CF₃ or —(CF₂)_(m)—F, wherem is hereinbefore defined;L is a linking group having a chain from 1-20 linked atoms selected fromthe group consisting of carbon, nitrogen, oxygen and sulphur atoms;M is a target bonding group;P is a conjugated component;the groups R¹⁵ are hydrogen or two or more of R¹⁵ groups are combined toform a one-ring or two-fused hydrocarbon ring system each ring havingfive or six carbon atoms, and remaining groups R¹⁵ are hydrogen; andn is an integer from 1 to 3;provided that:

-   i) at least one of groups R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰    R¹¹, R¹², R¹³ and R¹⁴ is -L-M or -L-P;-   ii) at least one of groups R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹ and R¹⁹    comprises fluorine; and-   iii) when any one of R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹ and R¹⁹ is —SO₃H at    least one of groups R¹¹, R¹², R¹³ and R¹⁴ is selected from -L-M,    -L-P and —(CH₂)_(k)—SO₃H.

The compounds according to the formula (I) will suitably include acounter-ion, which may be positive or negative to balance the formalcharge (or charges) on the dye chromophore. The nature of thecounter-ion is not material to the invention and could be one of manyknown ions such as NH₄ ⁺, K⁺, Na⁺, trifluoroacetate (F₃C—CO₂ ⁻),perchlorate (ClO₄ ⁻), Br⁻, or I⁻. In the context of the presentinvention, it is to be understood that the sulphonic acid group (—SO₃H)will also include the sulphonate group (—SO₃ ⁻), since sulphonate is theionised form of the parent acid.

In one embodiment according to the first aspect, compounds of formula(I) are provided wherein:

the groups R¹ and R² are selected independently from -L-M, -L-P, C₁-C₆alkyl and —(CH₂)_(k)—SO₃H;the groups R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹ and R¹⁹ are selected independentlyfrom hydrogen, —SO₃H, —SO₂—CF₃ and —(CF₂), —F, where m is 0 or aninteger from 1 to 4; or R³ taken in combination with R⁴ or R⁵ taken incombination with R⁶ and/or R⁷ taken in combination with R⁸ or R⁹ takenin combination with R¹⁹ form a fused aromatic six-membered ringcontaining carbon atoms and being optionally substituted one or moretimes by —SO₃H, —SO₂—CF₃ or —(CF₂), —F, where m is hereinbefore defined;the groups R¹¹, R¹², R¹³ and R¹⁴ are selected independently from C₁-C₆alkyl and —(CH₂)_(k)—SO₃H; andgroups R¹⁵, L, M and P, k and n are hereinbefore defined;provided that:

-   i) at least one of groups R¹ and R² is -L-M or -L-P;-   ii) at least one of groups R³, R⁴, R⁵, R⁶, R⁷, R⁸, m R⁹ and R¹⁰    comprises fluorine; and-   iii) when any one of R³, R⁴, R⁵, R⁶, R⁷, R⁸, m R⁹ and R¹⁰ is —SO₃H    at least one of groups R¹¹, R¹², R¹³ and R¹⁴ is —(CH₂)_(k)—SO₃H.

In an alternative embodiment according to the first aspect, compounds offormula (I) are provided wherein:

the groups R¹¹, R¹², R¹³ and R¹⁴ are selected independently from -L-M,-L-P, C₆ alkyl and —(CH₂)_(k)—SO₃H,the groups R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹ and R¹⁰ are selected independentlyfrom hydrogen, —SO₃H, —SO₂—CF₃ and —(CF₂), —F, where m is 0 or aninteger from 1 to 4; or R³ taken in combination with R⁴ or R⁵ taken incombination with R⁶ and/or R⁷ taken in combination with R⁸ or R⁹ takenin combination with R¹⁰ form a fused aromatic six-membered ringcontaining carbon atoms and being optionally substituted one or moretimes by —SO₃H, —SO₂—CF₃ or —(CF₂), —F, where m is hereinbefore defined;the groups R¹ and R² are selected independently from C₁-C₆ alkyl and—(CH₂)_(k)—SO₃H; andgroups R¹⁵, L, M and P, k and n are hereinbefore defined;provided that:

-   i) at least one of groups R¹¹, R¹², R¹³ and R¹⁴ is -L-M or -L-P;-   ii) at least one of groups R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹ and R¹⁰    comprises fluorine; and-   iii) when any one of R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹ and R¹⁰ is —SO₃H at    least and R¹⁴ is —(CH₂)_(k)—SO₃H.

Preferably, at least one of groups R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹ and R¹⁰ isfluorine.

In one preferred embodiment, groups R¹⁵ are all hydrogen. In analternative embodiment, the polymethine bridge linking the heterocyclicportions of the compounds according to formula (I) may comprise ahydrocarbon ring system of general formula:

wherein n is an integer from 1 to 3 and p is 0 or 1. Any remaininggroups R¹⁵ are hydrogen. Examples of hydrocarbon ring systems formingthe polymethine bridge are shown in Table 1.

TABLE 1

The linking group L links the target bonding group M or conjugatedcomponent P with the cyanine chromophore in the compounds according toformula (I) and is suitably covalently attached to the R¹, R², R³, R⁴,R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³ and/or R¹⁴ positions of the dye.Preferably, the dyes of the present invention contain a single targetbonding group -L-M, covalently attached to one of the R¹, R², R¹¹, R¹²,R¹³ or R¹⁴ positions. The skilled person will understand that dyesaccording to the present invention and having a target bonding group mayalso include the group -L-P, wherein the fluorescent dye is conjugatedto a target component. L is suitably a straight or branched chain offrom 1 to 20 linked atoms containing carbon, nitrogen, oxygen andsulphur atoms. Preferably, L is a linking group having the formula:

—(CHR′)_(p)-Q-(CHR′)_(r)—

where Q is selected from: —CHR′, —NR′—, —O—, —S—, —CR′═CR′—, —Ar—,—C(O)—NR′— and —C(O)—O—, where R′ is hydrogen or C₁-C₄ alkyl; Ar isphenylene, optionally substituted with sulphonate; p is 0-5 and r is1-5. Q is preferably selected from: —CHR′— and —C(O)—NH—, where R′ ishydrogen or C₁-C₄ alkyl; more preferably Q is —CHR′— and R′ is hydrogen.In particularly preferred embodiments according to the invention, -L-Mand/or -L-P comprise a carboxypentyl group.

Suitably, the fluorescent cyanine dyes of the present invention compriseat least one, preferably two or more fluorine atoms substituted directlyor indirectly onto the cyanine dye chromophore. In one embodiment,compounds of formula (I) are suitably substituted by a fluorine atom atleast one, preferably at least two, and more preferably at least threeof the R³, R⁴, R⁵ and R⁶ positions and/or the R⁷, R⁸, R⁹ and R¹⁹positions. In this embodiment, substitution by one or more fluorineatoms may give rise to symmetric or asymmetric dyes. In particularlypreferred embodiments, each of the R³, R⁴, R⁵ and R⁶ positions and/orthe R⁷, R⁸, R⁹ and R¹⁹ positions are substituted by fluorine. Perfluorosubstitution of the cyanine dye chromophore has been found to lowerdye-dye aggregation, thereby reducing fluorescence quenching andenhancing dye photostability (Waggoner, A. et al, loc cit).Alternatively, the compounds of formula (I) may include a perfluoroC₁-C₄ alkyl substituent at one, preferably not more than two of the R³,R⁴, R⁵ or R⁶ positions and/or the R⁷, R⁸, R⁹ or R¹⁹ positions. Anyremaining groups R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹ and R¹⁰ are selected from Hor F, provided that at least one of the remaining groups R³, R⁴, R⁵, R⁶,R⁷, R⁸, R⁹ and R¹⁰ is F. Preferably, the perfluoro C₁-C₄ alkylsubstituent is trifluoromethyl. In a still further embodiment, thecompounds of formula (I) may include the group —SO₂—CF₃ substituted atleast one, usually not more than two of the R³, R⁴, R⁵ or R⁶ positionsand/or the R⁷, R⁸, R⁹ or R¹⁹ positions. Remaining groups R³, R⁴, R⁵, R⁶,R⁷, R⁹ and R¹⁰ are selected from H or F, preferably F.

Particular examples of cyanine dyes according to the present inventionhaving indole or benzindole ring systems are shown as compounds offormula (II), (III) and (IV) in Table 2.

TABLE 2

(II)

(III)

(IV)

In the compounds of formula (II), (III) and (IV), at least one of groupsR¹, R², R¹¹, R¹², R¹³ and R¹⁴ is -L-M or -L-P, where L is a linkinggroup having the formula:

—(CHR)_(p)-Q-(CHR′)_(r)—

where Q is selected from: —CHR′—, —NR′—, —O—, —S—, —C(O)—NR′— and—C(O)—O—, where R′ is hydrogen or C₁-C₄ alkyl; p is 0-5 and r is 1-5;M is a target bonding group; andP is a conjugated component; is not -L-M or -L-P, said remaining groupsR¹, R², R¹¹, R¹², R¹³ and R¹⁴ are selected independently from C₁-C₆alkyl, and —(CH₂)_(k)—SO₃H, where k is an integer from 1 to 10; at leastone of groups R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹ and R¹⁰ comprises at least onefluoro group selected from —SO₂—CF₃ and —(CF₂)_(m)—F, where m is 0 or aninteger from 1 to 4;when any of groups R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹ and R¹⁰ is not —SO₂—CF₃ or—(CF₂)_(m)—F, remaining groups R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹ and R¹⁰ arehydrogen; groups R¹⁶, R¹⁷, R¹⁸, R¹⁹, R²⁰, R²¹, R²² and R²³ when presentare selected independently from hydrogen, —SO₂—CF₃ and —(CF₂)_(m)—F,where m is hereinbefore defined; andn is an integer from 1 to 3.

In one embodiment, at least one of groups R¹ and R² of the compounds offormula (II), (III) and (IV) is -L-M or -L-P and any remaining group R¹or R² is selected from C₁-C₆ alkyl and —(CH₂)_(k)—SO₃H;

at least one of groups R¹¹, R¹², R¹³ and R¹⁴ is —(CH₂)_(k)—SO₃H and anyremaining groups R¹¹, R¹², R¹³ and R¹⁴ are C₁-C₆ alkyl; andgroups R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹⁶, R¹⁷, R¹⁸, R¹⁹, R²⁰, R²¹,R²², R²³, L, M, P, k and n are hereinbefore defined. Preferably,remaining group R¹ or R² is —(CH₂)_(k)—SO₃H.

Preferably, at least one of groups R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹ and R¹⁰ isfluorine.

In this embodiment, suitably, at least two of groups R¹¹, R¹², R¹³ andR¹⁴ are the group —(CH₂)_(k)—SO₃H. Preferably, one of groups R¹¹ andR¹², and one of groups R¹³ and R¹⁴ is the group —(CH₂)_(k)—SO₃H, andremaining groups R¹¹, R¹², R¹³ or R¹⁴ are C₁-C₆ alkyl, where k ishereinbefore defined. In these embodiments, remaining groups R¹¹ or R¹²and R¹³ or R¹⁴ are preferably methyl. Preferably k is 3 or 4. Thus,particularly preferred —(CH₂)_(k)—SO₃H groups are selected from—(CH₂)₃—SO₃H and —(CH₂)₄—SO₃H.

In an alternative embodiment, at least one of groups R¹¹, R¹², R¹³ andR¹⁴ of the compounds of formula (II), (III) and (IV) is -L-M or -L-P andany remaining groups R¹¹, R¹², R¹³ and R¹⁴ are selected from C₁-C₆alkyl, and —(CH₂)_(k)—SO₃H; at least one of groups R¹ and R² is—(CH₂)_(k)—SO₃H and any remaining group R¹ or R² is C₁-C₆ alkyl; and

groups R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹⁶, R¹⁷, R¹⁸, R¹⁹, R²⁰, R²¹,R²², R²³, L, M, P, k and n are hereinbefore defined.

Preferably, at least one of groups R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹ and R¹⁰ isfluorine.

Preferably, in this embodiment, both groups R¹ and R² are—(CH₂)_(k)—SO₃H, where k is an integer from 1 to 10. Particularlypreferred groups —(CH₂)_(k)—SO₃H are those in which k is 3 or 4, that is—(CH₂)₃—SO₃H and —(CH₂)₄—SO₃H.

Preferably, Q is selected from —CHR′— and —C(O)—NH—, where R′ ishydrogen or C₁-C₄ alkyl. More preferably Q is —CHR′— and R′ is hydrogen.In particularly preferred embodiments, -L-M and/or -L-P comprise acarboxypentyl group.

In structures (II) to (IV), at least one of groups R³, R⁴, R⁵, R⁶, R⁷,R⁸, R⁹ and R¹⁰ comprises at least one fluoro group selected from—SO₂—CF₃ and —(CF₂)_(m)—F, where m is 0 or an integer from 1 to 4. Anyremaining groups R³, R⁴, R⁵, R⁶, R⁸, R⁹ and R¹⁹ are hydrogen. Suitably,at least one, preferably at least two, and more preferably at leastthree of the R³, R⁴, R⁵ and R⁶ positions and/or the R⁷, R⁸, R⁹ and R¹⁹positions are substituted by fluorine. In particularly preferredembodiments, each of the R³, R⁴, R⁵ and R⁶ positions and/or the R⁷, R⁸,R⁹ and R¹⁰ positions are substituted by fluorine and any remaininggroups R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹ and R¹⁰ are hydrogen. In alternativeembodiments, at least one and usually not more that two of groups R³,R⁴, R⁵ and R⁶ and/or groups R⁷, R⁸, R⁹ and R¹⁰ is a perfluoro C₁-C₄alkyl group or a —SO₂—CF₃ group. Any remaining groups R³, R⁴, R⁵, R⁶,R⁷, R⁸, R⁹ and R¹⁰ are selected from H or F, preferably F. A preferredperfluoro C₁-C₄ alkyl group is trifluoromethyl.

Groups R¹⁶, R¹⁷, R¹⁸, R¹⁹, R²⁰, R²¹, R²² and R²³ when present areselected independently from hydrogen, —SO₂—CF₃ and —(CF₂), —F, wherein mis hereinbefore defined. Usually not more than two, preferably not morethan one —SO₂—CF₃ or perfluoro —C₄ alkyl group is substituted at theR¹⁶, R¹⁷, R¹⁸, R¹⁹ and/or R²⁰, R²¹, R²² and positions. Preferably,groups R¹⁶, R¹⁷, R¹⁸, R¹⁹, R²⁰, R²¹, R²² and R²³ are selectedindependently from hydrogen and fluorine.

Optionally, dyes according to the present invention having 1, 2, 3, or 4fluoro groups attached thereto, may be further substituted with one ormore sulphonic acid groups attached directly to the remaining R³, R⁴,R⁵, R⁶, R⁷, R⁸, R⁹ or R¹⁰ positions. Thus, dyes according to the presentinvention may be substituted directly or indirectly with up to four ormore sulphonic acid groups, preferably between three and five sulphonicacid groups. The use of cyanine dyes substituted by fluorine and havingadditional substitution with three or more sulphonic acid groups forlabelling biological target molecules results in a labelled product inwhich there is reduced dye-dye aggregation and improved photostability,compared with cyanines having no such substitutions. The fluorescenceemission intensity of a molecule so labelled with the preferred dyes ofthe present invention increases with the number of covalently attacheddyes. Furthermore, substitution of the indolinium 1- and/or 3-positionswith sulphonic acid groups in addition to increasing the overall chargeon the dye molecule, also adds steric bulk, thereby contributing to areduction in dye-dye aggregation.

Suitably, the target bonding group M is a group that reacts with acomplementary group of a component to be labelled, with the formation ofa covalent linkage between the dye and the conjugated component. Thechoice of target bonding group will depend on the groups that areavailable on the component to be labelled and, as such, will be wellknown to the skilled person. For example, the target bonding group maybe a reactive group that can react under suitable conditions with acomplementary functional group of a component. Examples of functionalgroups present in components, such as proteins, peptides, nucleic acidscarbohydrates and the like, include hydroxy, amino, sulphydryl, carbonyl(including aldehyde and ketone), carboxylic acid and thiophosphate.Alternatively, the target bonding group M may be a functional group andthe target may contain, or be derivatised to contain a reactiveconstituent, such that the functional group of the dye may be reactedunder suitable conditions with the reactive group of the targetcomponent. In either case, the component becomes labelled with the dyeaccording to formula (I). Suitably, when M is a reactive group, it isselected from succinimidyl ester, sulpho-succinimidyl ester,4-sulfo-2,3,5,6-tetrafluorophenol (STP) ester, isothiocyanate,maleimide, haloacetamide, acid halide, hydrazide, vinylsulphone,dichlorotriazine and phosphoramidite. Preferably, the reactive group isa succinimidyl ester of a carboxylic acid, an isothiocyanate, amaleimide, a haloacetamide or a phosphoramidite. When M is a functionalgroup, it is suitably selected from hydroxy, amino, sulphydryl, carbonyl(including aldehyde and ketone), carboxylic acid and thiophosphate,preferably hydroxy, amino or sulphydryl. By virtue of these reactive andfunctional groups the compounds of formula (I) may be reacted with andbecome covalently bound to the target component.

Examples of reactive groups at the R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹,R¹⁰, R¹¹, R¹², R¹³ and/or R¹⁴ positions of the compound according toformula (I) and the groups with which groups R¹, R², R³, R⁴, R⁵, R⁶, R⁷,R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³ and/or R¹⁴ can react are provided in Table 3.In the alternative, R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹²,R¹³ and/or R¹⁴ may be the functional groups of Table 3 which would reactwith the reactive groups of a target component.

TABLE 3 Reactive Groups and Functional Groups Reactive TherewithReactive Groups Functional Groups succinimidyl ester, sulpho- primaryamino, secondary amino succinimidyl ester4-sulfo-2,3,5,6-tetrafluorophenol primary amino, secondary amino (STP)ester anhydrides, acid halides primary amino, secondary amino, hydroxylisothiocyanate amino groups vinylsulphone amino groups dichlorotriazinesamino groups haloacetamides, maleimides thiols, imidazoles, hydroxyl,amines, thiophosphates carbodiimide carboxylic acids hydrazine,hydrazide carbonyl including aldehyde and ketone phosphoramiditeshydroxyl groups

Particularly preferred reactive groups which are especially useful forlabelling target components with available amino and hydroxyl functionalgroups include:

Particularly preferred reactive groups which are useful for labellingtarget components with available thiol functional groups include:

Particularly preferred examples of the group -L-M are those whichcomprise a carboxypentyl group, for example:

In another embodiment, the target bonding group M may be an affinity tagwhich is capable of binding specifically and non-covalently with itscomplementary specific binding partner, thereby forming a specificbinding pair. Examples of specific binding pairs include, but are notrestricted to biotin/avidin, biotin/streptavidin, polyhistidinetag-metal ion complexes with nitrilotriacetic acid (e.g. Ni²⁺: NTA). Thecomplementary specific binding partner may be one component of alabelling complex for detection of a target component. Thus, in onepreferred labelling format, streptavidin, having four sites ofattachment for a biotin label, may be used as a bridge linking a biotingroup on the target component with a dye according to the presentinvention wherein group M is biotin, iminobiotin or desthiobiotin. It isto be understood that in the context of the present invention, any twoatoms or molecules that possess a specific binding affinity one for theother, may be employed. Preferred examples of affinity tags are selectedfrom biotin, iminobiotin and desthiobiotin.

The following are more specific examples of cyanine dyes according tothe invention, as shown in Table 4.

TABLE 4

(II)

(III)

(IV)wherein:groups R¹, R² and R¹¹ are selected from —(CH₂)₅—COOH and —(CH₂)₄—SO₃H;group R¹³ is selected from —(CH₂)₅—COOH, —(CH₂)₄—SO₃H and —CH₃; groupsR¹² and R¹⁴ are —CH₃;at least two of groups R³, R⁴, R⁵ and R⁶ and/or groups R⁷, R⁸, R⁹ andR¹⁹ are F; remaining groups R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹ or R¹⁹ areselected from H, —SO₃, —SO₂—CF₃ and —CF₃;groups R¹⁶, R¹⁷, R¹⁸ and R¹⁹ and/or groups R²⁹, R²¹, R²² and R²² whenpresent are F; andn is an integer from 1 to 3.

In compounds of the present invention, group -L-M is suitably asuccinimidyl ester derivative of an alkyl carboxylic acid, preferably5-carboxypentyl, N-hydroxysuccinimidyl ester, or 5-carboxypentyl,N-hydroxy-sulphosuccinimidyl ester.

Particular examples of dyes according to the first aspect of theinvention are as follows:

-   i)    2-{(1E,3E,5E)-5-[1-(5-Carboxypentyl)-4,5,6,7-tetrafluoro-3-methyl-3-(4-sulfobutyl)-1,3-dihydro-2H-indol-2-ylidene]penta-1,3-dienyl}-4,5,6,7-tetrafluoro-3-methyl-1,3-bis(4-sulfobutyl)-3H-indolium    (Cy5F) (Compound 1);-   ii)    2-{(1E,3E,5E,7E)-7-[1-(5-Carboxypentyl)-4,5,6,7-tetrafluoro-3-methyl-3-(4-sulfobutyl)-1,3-dihydro-2H-indol-2-ylidene]hepta-1,3-5-trienyl}-4,5,6,7-tetrafluoro-3-methyl-1,3-bis(4-sulfobutyl)-3H-indolium    (Cy7F) (Compound 2);-   iii)    3-(5-Carboxypentyl)-3-methyl-5-sulfo-1-(4-sulfobutyl)-2-{1E,3E,5E}-5-[4,5,6,7-tetrafluoro-3-methyl-1,3-bis(4-sulfobutyl)-1,3-dihydro-2H-indol-2-ylidene]penta-1,3-dienyl}-3H-indolium    (Compound 3);-   iv)    1-(5-Carboxypentyl)-3-methyl-5-sulfo-3-(4-sulfobutyl)-2-{(1E,3E,5E)-5-[4,5,6,7-tetrafluoro-3-methyl-1,3-bis(4-sulfobutyl)-1,3-dihydro-2H-indol-2-ylidene]penta-1,3-dienyl}-3H-indolium    (Compound 4);-   v)    2-{(1E,3E,5E)-5-[1-(5-Carboxypentyl)-4,5,6,7-tetrafluoro-3-methyl-3-(4-sulfobutyl)-1,3-dihydro-2H-indol-2-ylidene]penta-1,3-dienyl}-4,6-difluoro-3-methyl-1,3-bis(4-sulfobutyl)-3H-indolium    (Compound 5);-   vi)    2-{(1E,3E,5E)-5-[1-(5-Carboxypentyl)-4,6-difluoro-3,3-dimethyl-1,3-dihydro-2H-indol-2-ylidene]penta-1,3-dienyl}-4,5,6,7-tetrafluoro-3-methyl-1,3-bis(4-sulfobutyl)-3H-indolium    (Compound 6);-   vii)    1-(5-Carboxypentyl)-2-{(1E,3E,5E)-5-[1-(5-carboxypentyl)-4,6-difluoro-3,3-dimethyl-1,3-dihydro-2H-indol-2-ylidene]penta-1,3-dienyl}-4,5,6,7-tetrafluoro-3-methyl-3-(4-sulfobutyl)-3H-indolium    (Compound 7);-   viii)    2-{(1E,3E,5E)-5-[1-(5-Carboxypentyl)-4,5,6,7-tetrafluoro-3-methyl-3-(4-sulfobutyl)-1,3-dihydro-2H-indol-2-ylidene]penta-1,3-dienyl}-3-methyl-1,3-bis(4-sulfobutyl)-5-[(trifluoromethyl)sulfonyl]-3H-indolium    (Compound 8);-   ix)    2-{(1E,3E,5E)-5-[1-(4-Sulfobutyl)-4,6-bis(trifluoromethyl)-3-methyl-3-(5-carboxypentyl))-1,3-dihydro-2H-indol-2-ylidene]penta-1,3-dienyl}-4,6-difluoro-3-methyl-1,3-bis(4-sulfobutyl)-3H-indolium    (Compound 9);-   x)    2-{(1E,3E,5E)-5-[1-(5-Carboxypentyl)-4,5,6,7-tetrafluoro-3-methyl-3-(4-sulfobutyl)-1,3-dihydro-2H-indol-2-ylidene]penta-1,3-dienyl}-3-methyl-1,3-bis(4-sulfobutyl)-6-(trifluoromethyl)-3H-indolium    (Compound 10);-   xi)    2-{(1E,3E,5E)-5-[1-(5-Carboxypentyl)-4,5,6,7-tetrafluoro-3-methyl-3-(4-sulfobutyl)-1,3-dihydro-2H-indol-2-ylidene]penta-1,3-dienyl}-4,5,6,7,8,9-hexafluoro-1,1-dimethyl-3-(4-sulfobutyl)-1H-benzo[e]indolium    (Compound 11);-   xii)    2-{(1E,3E,5E)-5-[1-(5-Carboxypentyl)-4,6-difluoro-3,3-dimethyl-1,3-dihydro-2H-indol-2-ylidene]penta-1,3-dienyl}-6-fluoro-3-methyl-1,3-bis(4-sulfobutyl)-4-(trifluoromethyl)-3H-indolium    (Compound 12);-   xiii)    2-{(1E,3E,5E)-5-[1-(5-Carboxypentyl)-4,6-difluoro-3,3-dimethyl-1,3-dihydro-2H-indol-2-ylidene]penta-1,3-dienyl}-4,6-difluoro-3-methyl-1,3-bis(4-sulfobutyl)-3H-indolium    (Compound 13);-   xiv)    2-{(1E,3E,5E)-5-[1-(5-Carboxypentyl)-4,6-difluoro-3,3-dimethyl-1,3-dihydro-2H-indol-2-ylidene]penta-1,3-dienyl}-3-methyl-5-sulfo-1,3-bis(4-sulfobutyl)-3H-indolium    (Compound 14);-   xv)    2-{(1E,3E,5E)-5-[3-(5-Carboxypentyl)-4,5,6,7-tetrafluoro-3-methyl-1-(4-sulfobutyl)-1,3-dihydro-2H-indol-2-ylidene]penta-1,3-dienyl}-4,5,6,7-tetrafluoro-3-methyl-1,3-bis-(4-sulfobutyl)-3H-indolium    (Compound 15);-   xvi)    2-{(1E,3E,5E)-5-[3-(5-Carboxypentyl)-6-fluoro-3-methyl-1-(4-sulfobutyl)-4-(trifluoromethyl)-1,3-dihydro-2H-indol-2-ylidene]penta-1,3-dienyl}-4,5,6,7-tetrafluoro-3-methyl-1,3-bis(4-sulfobutyl)-3H-indolium    (Compound 16);-   xvii)    2-{(1E,3E,5E)-5-[3-(5-Carboxypentyl)-4-trifluoromethyl-6-fluoro-3-methyl-1,3-dihydro-2H-indol-2-ylidene]penta-1,3-dienyl}-3-(5-carboxypentyl)-4-trifluoromethyl-6-fluoro-3-methyl-3H-indolium    (Compound 17);-   xviii)    2-{(1E,3E,5E,7E)-7-[1-(5-Carboxypentyl)-4,5,6,7-tetrafluoro-3-methyl-3-(4-sulfobutyl)-1,3-dihydro-2H-indol-2-ylidene]hepta-1,3,5-trienyl}-3-methyl-1,3-bis(4-sulfobutyl)-4,6-bis(trifluoromethyl)-3H-indolium    (Compound 18);-   xix)    2-{(1E,3E,5E,7E)-7-[1-(5-Carboxypentyl)-4,5,6,7-tetrafluoro-3-methyl-3-(4-sulfobutyl)-1,3-dihydro-2H-indol-2-ylidene]hepta-1,3,5-trienyl}-3-methyl-1,3-bis(4-sulfobutyl)-5-trifluoromethyl-3H-indolium    (Compound 19);-   xx)    2-{(1E,3E)-3-[1-(5-Carboxypentyl)-4,5,6,7-tetrafluoro-3-methyl-3-(4-sulfobutyl)-1,3-dihydro-2H-indol-2-ylidene]prop-1-enyl}-4,5,6,7-tetrafluoro-3-methyl-1,3-bis(4-sulfobutyl)-3H-indolium    (Compound 20); and-   xxi)    1-(5-Carboxypentyl)-2-{(1E,3E)-3-[1-(5-carboxypentyl)-4,5,6,7-tetrafluoro-3-methyl-3-(4-sulfobutyl)-1,3-dihydro-2H-indol-2-ylidene]prop-1-enyl}-4,5,6,7-tetrafluoro-3-methyl-3-(4-sulfobutyl)-3H-indolium    (Compound 21).

The present invention also relates to labelling methods wherein thecompounds according to the first aspect including at least one group-L-M attached to the R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹²,R¹³ and/or R¹⁴ positions as hereinbefore defined may be used to labeland thereby impart fluorescent properties to a target component. Inparticular, they may be used for multiple labelling and detection ofbiological molecules, such as nucleic acids, DNA, RNA, oligonucleotides,nucleotides, proteins, peptides, antibodies, etc. Thus, in a secondaspect, there is provided a method for covalently labelling a component,the method comprising:

-   a) contacting said component with a compound of formula (I):

wherein:the groups R¹, R², R¹¹, R¹², R¹³ and R¹⁴ are selected independently from-L-M, -L-P, C₁-C₆ alkyl and —(CH₂)_(k)—SO₃H, where k is an integer from1 to 10; the groups R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹ and R¹⁰ are selectedindependently from hydrogen, -L-M, -L-P, —SO₃H, —SO₂—CF₃ and—(CF₂)_(m)—F, where m is 0 or an integer from 1 to 4; or R³ taken incombination with R⁴ or R⁵ taken in combination with R⁶ and/or R⁷ takenin combination with R⁸ or R⁹ taken in combination with R¹⁰ form a fusedaromatic six-membered ring containing carbon atoms and being optionallysubstituted one or more times by —SO₃H, —SO₂—CF₃ or —(CF₂)_(m)—F, wherem is hereinbefore defined;L is a linking group having a chain from 1-20 linked atoms selected fromthe group consisting of carbon, nitrogen, oxygen and sulphur atoms;M is a target bonding group;P is a conjugated component;the groups R¹⁵ are hydrogen or two or more of R¹⁵ groups are combined toform a one-ring or two-fused hydrocarbon ring system each ring havingfive or six carbon atoms, and remaining groups R¹⁵ are hydrogen; andn is an integer from 1 to 3;provided that:

-   i) at least one of groups R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰    R¹¹, R¹², R¹³ and R¹⁴ is -L-M;-   ii) at least one of groups R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹ and R¹⁰    comprises fluorine; and-   iii) when any one of R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹ and R¹⁰ is —SO₃H at    least one of groups R¹¹, R¹², R¹³ and R¹⁴ is selected from -L-M and    —(CH₂)_(k)—SO₃H; and-   b) reacting said compound with said component such that said    compound labels said component.

Preferably, at least one of groups R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹ and R¹⁰ isfluorine.

Preferably, groups R¹⁵ are hydrogen.

The target bonding group M is a group suitable for the formation of acovalent link between the compound of formula (I) and the targetcomponent, such as a reactive or functional group as hereinbeforedefined. The method comprises incubating the component to be labelledwith an amount of the compound according to the invention underconditions such that the dye becomes covalently bound to the component.Methods for the formation of dye conjugates or complexes with targetcomponents will be well known to the skilled person. For example,covalent labelling of proteins is typically performed in an aqueousbuffered medium, suitably bicarbonate at pH 9.0, at ambient temperaturefor a period of typically 1 hour. The reaction is normally carried outin the dark. The labelled protein can be separated from any unreacteddye by size exclusion chromatography, for example using Sephadex™ as thestationary phase and phosphate buffer, pH 7.0 as the eluant. Formultiple labelling of a target biomolecule, the ratio of the amount orconcentration of dye to target material should be adjusted accordingly.

Suitable target components may include, but are not limited to the groupconsisting of antibody, lipid, protein, peptide, carbohydrate,nucleotides which contain or are derivatized to contain one or more ofan amino, sulphydryl, carbonyl, hydroxyl, carboxylic acid andthiophosphate groups, and oxy or deoxy polynucleic acids which containor are derivatized to contain one or more of an amino, sulphydryl,carbonyl, hydroxyl, carboxylic acid and thiophosphate groups, microbialmaterials, drugs, hormones, cells, cell membranes, toxins, polymerparticles, and glass beads.

Components, for example proteins, labelled with the fluorescent dyes ofthe present invention may be used in in vitro assays to monitor cellularevents, for example, uptake, transport, receptor binding, metabolism,signal transduction and protein-protein interactions, or to visualizecellular and molecular events in vivo. The dye-labelled components maybe detected either directly or indirectly by an optical imagingprocedure, for example using charge coupled device (CCD) detection.Suitable high-throughput sub-cellular imaging instrumentation isavailable, for example IN Cell Analyser 1000 and eXplore Optix™, In vivoFluorescence Optical Imaging System (GE Healthcare). eXplore Optix™ is atrademark of ART Advanced Research Technologies Inc.

In addition to the foregoing one-step labelling process, the presentinvention also relates to two-step labelling processes in which, in afirst step, a dye according to the present invention binds to, andthereby labels a primary component, such as an antibody, protein, DNAprobe, etc. In the second step of the labelling process, thefluorescently labelled primary component is then used as a probe fordetection of a secondary component, such as an antigen for which theantibody is specific.

The compounds of the present invention can also be used to determine theconcentration of a particular protein or other component in a system. Ifthe number of reactive groups on a protein which can react with a probeis known, the fluorescence per molecule can be known and theconcentration of these molecules in the system can be determined by thetotal fluorescence intensity of the system. This particular method canbe used to measure the concentration of various labelled analytes usingmicrotitre plate readers or other known immunofluorescence detectionsystems. The concentration of fluorescently labelled material can alsobe determined using, for example, fluorescence polarization detectioninstruments.

The compounds of the present invention may also be used in a detectionmethod wherein a plurality of the fluorescent dyes are covalentlyattached to a plurality of different primary components, such asantibodies, each primary component being specific for a differentsecondary component, such as an antigen, in order to identify each of aplurality of secondary components in a mixture of secondary components.According to this method of use, each of the primary components isseparately labelled with a fluorescent dye having a different lightabsorption and emission wavelength characteristic, compared with the dyemolecules used for labelling the other primary components. The labelledprimary components are then added to the preparation containingsecondary components, such as antigens, and the primary components areallowed to attach to the respective secondary components for which theyare selective.

Any unreacted probe materials may be removed from the preparation by,for example, washing, to prevent interference with the analysis. Thepreparation is then subjected to a range of excitation wavelengthsincluding the absorption wavelengths of particular fluorescentcompounds. A fluorescence microscope or other fluorescence detectionsystem, such as a flow cytometer or fluorescence spectrophotometer,having filters or monochromators to select the rays of the excitationwavelength and to select the wavelengths of fluorescence is nextemployed to determined the intensity of the emission wavelengthscorresponding to the fluorescent compounds utilized, the intensity offluorescence indicating the quantity of the secondary component whichhas been bound with a particular labelled primary component. Knowntechniques for conducting multi-parameter fluorescence studies include,for example, multiparameter flow cytometry. In certain cases a singlewavelength of excitation can be used to excite fluorescence from two ormore materials in a mixture where each fluoresces at a differentwavelength and the quantity of each labelled species can be measured bydetecting its individual fluorescence intensity at its respectiveemission wavelength. If desired, a light absorption method can also beemployed.

The detection method of the present invention can be applied to anysystem in which the creation of a fluorescent primary component ispossible. For example, an appropriately reactive fluorescent compoundcan be conjugated to a DNA or RNA fragment and the resultant conjugatethen caused to bind to a complementary target strand of DNA or RNA.Appropriate fluorescence detection equipment can then be employed todetect the presence of bound fluorescent conjugates.

The present invention relates to intermediates and to methods suitablefor preparing the dyes of formula (I). Thus, in a third aspect, there isprovided a compound of formula (A):

wherein:the groups R¹, R¹¹ and R¹² are selected independently from -L-M, -L-P,C₁-C₆ alkyl and —(CH₂)_(k)—SO₃H, where k is an integer from 1 to 10; thegroups R³, R⁴, R⁵ and R⁶ are selected independently from hydrogen, -L-M,-L-P, —SO₃H, —SO₂—CF₃ and —(CF₂)_(m)—F, where m is 0 or an integer from1 to 4; or R³ taken in combination with R⁴ or R⁵ taken in combinationwith R⁶ form a fused aromatic six-membered ring containing carbon atomsand being optionally substituted one or more times by —SO₃H, —SO₂—CF₃ or—(CF₂)_(m)—F, where m is hereinbefore defined;L is a linking group having a chain from 1-20 linked atoms selected fromthe group consisting of carbon, nitrogen, oxygen and sulphur atoms;M is a target bonding group; andP is a conjugated component;provided that:

-   i) at least one of groups R¹, R³, R⁴, R⁵, R⁶, R¹¹ and R¹² is -L-M or    -L-P;-   ii) at least one of groups R³, R⁴, R⁵ and R⁶ comprises fluorine; and-   iii) when any one of R³, R⁴, R⁵ and R⁶ is —SO₃H at least one of    groups R¹¹ and R¹² is selected from -L-M, -L-P and —(CH₂)_(k)—SO₃H.

In one embodiment, at least one of groups R¹, R¹¹ and R¹² is -L-M, whereL is hereinbefore defined and M comprises a reactive group selected fromsuccinimidyl ester, sulpho-succinimidyl ester,4-sulfo-2,3,5,6-tetrafluorophenol (STP) ester, isothiocyanate,maleimide, haloacetamide, acid halide, hydrazide, vinylsulphone,dichlorotriazine and phosphoramidite.

In another embodiment, at least one of groups R¹, R¹¹ and R¹² is -L-M,where L is hereinbefore defined and M comprises a functional groupselected from hydroxy, amino, sulphydryl, imidazole, carbonyl includingaldehyde and ketone, carboxylic acid and thiophosphate.

Preferably, remaining groups R¹, R¹¹ and R¹² are selected C₁-C₆ alkyland —(CH₂)_(k)—SO₃H, where k is an integer from 1 to 10. Preferably k is3 or 4.

Preferably, at least one of groups R³, R⁴, R⁵ and R⁶ is fluorine. Morepreferably, at least two of groups R³, R⁴, R⁵ and R⁶ are F and anyremaining groups R³, R⁴, R⁵ or R⁶ are selected from H, —SO₂—CF₃ and CF₃,preferably H. In particularly preferred embodiments, groups R³, R⁴, R⁵and R⁶ are F.

Particular examples of compounds of formula (A) are those wherein:

at least one groups R¹ and R¹¹ is —(CH₂)_(k)—SO₃H;remaining group R¹ or R¹¹ is selected from —(CH₂)₅—COOH and—(CH₂)_(k)—SO₃H; group R¹² is C₁-C₆ alkyl, preferably methyl; groups R³,R⁴, R⁵ and R⁶ are F; andk is 3 or 4.

Compounds according to the invention may be prepared by a processcomprising:

-   a) reacting a first compound having the formula (A):

and

-   b) a second compound which may be the same or different from the    first compound and having the formula (B):

and

-   c) a third compound (C) under conditions suitable for forming a    conjugated linkage between said first and second compounds;    wherein:    the groups R¹, R², R¹¹, R¹², R¹³ and R¹⁴ are selected independently    from -L-M, -L-P, C₁-C₆ alkyl and —(CH₂)_(k)—SO₃H, where k is an    integer from 1 to 10;    the groups R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹ and R¹⁰ are selected    independently from hydrogen, -L-M, -L-P, —SO₃H, —SO₂—CF₃ and    —(CF₂)_(m)—F, where m is 0 or an integer from 1 to 4; or R³ taken in    combination with R⁴ or R⁵ taken in combination with R⁶ and/or R⁷    taken in combination with R⁸ or R⁹ taken in combination with R¹⁰    form a fused aromatic six-membered ring containing carbon atoms and    being optionally substituted one or more times by —SO₃H, —SO₂—CF₃ or    —(CF₂)_(m)—F, where m is hereinbefore defined;    L is a linking group having a chain from 1-20 linked atoms selected    from the group consisting of carbon, nitrogen, oxygen and sulphur    atoms;    M is a target bonding group; and    P is a conjugated component;    provided that:-   i) at least one of groups R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰,    R¹¹, R¹², R¹³ and R¹⁴ is -L-M or -L-P;-   ii) at least one of groups R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹ and R¹⁰    comprises fluorine; and-   iii) when any one of R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹ and R¹⁰ is —SO₃H at    least one of groups R¹¹, R¹², R¹³ and R¹⁴ is selected from -L-M,    -L-P and —(CH₂)_(k)—SO₃H.

Preferably, at least one of groups R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹ and R¹⁰ isfluorine.

Preferably, —(CH₂)_(k)—SO₃H is selected from —(CH₂)₃—SO₃H and—(CH₂)₄—SO₃H.

According to the method, intermediate compounds (A), (C) and (B) may bereacted either in a single step or in a multiple step process to formthe compounds of formula (I). Symmetrical compounds of formula (I)wherein structures (A) and (B) are the same may be suitably prepared byreacting a compound of formula (A) (or (B) in two molar proportions withan appropriate bis-functional methine fragment containing 1, 3 or 5carbon atoms, substituted with a group to form R¹⁵ as hereinbeforedefined. For example, a substituted N,N′-diphenylformamidine, or orthoester will be employed as the third compound (C) for preparingtrimethine cyanine dye analogues. In a corresponding manner, a suitablysubstituted malondialdehyde dianil may be employed for preparing thepentamethine cyanine dye analogues and a glutaconic aldehyde forpreparing heptamethine cyanine dye analogues. The reaction is usuallycarried out in an organic solvent, such as pyridine and heated toreflux. The mixture subsequently is cooled and poured into an organicsolvent such as ether. The resulting solid or semi-solid may be purifiedby chromatography on a silica gel column using a series ofmethanol/chloroform solvents. Alternatively, purification may beachieved using a reverse phase column with an eluant consisting of watercontaining 0.1% TFA/acetonitrile.

Unsymmetrical compounds of formula (I) wherein structures (A) and (B)are different may be conveniently prepared in a two step process. Inthis process, an intermediate compound is first formed by reacting anindolinium compound of formula (A) with a compound suitable for formingthe linkage, for example, a suitably substitutedN,N′-diphenylformamidine, or malonaldehyde dianil, in the presence ofacetic anhydride, to form a 2-anilinovinyl or 4-anilino-1,3-butadienylquaternary salt. The intermediate quaternary salt may be reacted with asecond 2-methyl indolinium quaternary salt to give a compound of formula(I). Alternative intermediates for forming the polymethine linkagejoining the heterocyclic ring systems are known and are described, forexample in Hamer, F. M., “The Cyanine Dyes and Related Compounds”,Interscience (1964).

EXAMPLES

Below, the present invention will be described by way of examples, whichare provided for illustrative purposes only and accordingly are not tobe construed as limiting the scope of the present invention as definedby the appended claims. All references given below and elsewhere in thisapplication are hereby included herein by reference.

1.2-{(1E,3E,5E)-5-[1-(5-Carboxypentyl)-4,5,6,7-tetrafluoro-3-methyl-3-(4-sulfobutyl)-1,3-dihydro-2H-indol-2-ylidene]penta-1,3-dienyl}-4,5,6,7-tetrafluoro-3-methyl-1,3-bis(4-sulfobutyl)-3H-indolium(Cy5F) (Compound 1)

1.1 5-(Ethoxycarbonyl)-5-methyl-6-oxoheptane-1-sulphonate, sodium salt

Sodium hydride (60 wt %, 12 g≡0.3 mol NaH) was slurried in dry DMF (100ml). The resulting suspension was cooled with stirring to 0° C. To thiswas added a solution of ethyl 2-methylacetoacetate (50 g, 0.346 mol) inDMF (25 ml), dropwise so as to maintain the temperature at <10° C. andcontrol effervescence. Once addition was complete and hydrogen evolutionceased, the mixture was warmed in a warm water bath until a clear, paleyellow solution resulted. This was cooled again to 0° C. A solution of1,4-butanesultone (45 g, 0.33 mol) in DMF (25 ml) was added over 15mins, maintaining the temperature at <10° C. Once addition was complete,the mixture was heated at 50° C. for 16 hrs. The solvent was thenevaporated under vacuum to dryness; the residue was partitioned betweenwater and diethyl ether. The aqueous layer was retained; the organiclayer was extracted with fresh water, then discarded. The combinedaqueous extracts were washed with fresh ether, then evaporated undervacuum to give the product as a waxy solid.

δH (270 MHz; D₂O) 4.23 (2H, q), 2.9 (2H, app t), 2.26 (3H, s), 2.0-1.6(6H, m), 1.36 (3H, s) and 1.26 (3H, t).

1.2 5-Methyl-6-oxoheptane-1-sulphonic acid

5-(Ethoxycarbonyl)-5-methyl-6-oxoheptane-1-sulphonate, sodium salt (from1.1) was heated at 90° C. in concentrated hydrochloric acid (200 ml),until TLC indicated complete reaction (˜3 hrs). The solvent was thenevaporated under vacuum; the residue was purified by flashchromatography (Silica. Ethanol/dichloromethane mixtures) to give 49.6 gof 5-methyl-6-oxoheptane-1-sulphonic acid.

δH (270 MHz; D₂O) 2.9 (2H, app t), 2.68 (1H, m), 2.2 (3H, s), 1.8-1.3(6H, m) and 1.18 (3H, d).

1.3 2,3-Dimethyl-3-(4-sulfobutyl)-4,5,6,7-tetrafluoro-3H-indole,disodium salt

2,3,4,5-Tetrafluoro-aniline (1.75 g, 0.01 M) was dissolved in conc. HCl(280 ml). The flask was maintained at −10° C. and a solution of NaNO₂ (1eq) in water (10 ml) added dropwise followed subsequently by a solutionof tin(II) chloride (3.4 g) in conc. HCl (40 ml). The reaction wasreturned to ambient temperature and stirred for 1 hr. The solvent wasremoved in vacuo to yield the crude product as a yellow salt (7 g).

The hydrazine salts and crude material were dissolved in acetic acid (50ml) with the sulfonated ketone, 5-methyl-6-oxoheptane-1-sulphonic acid(6 g). The solution was heated at 140° C. for 2 hrs to yield an orangesolution with fine orange precipitate. The solvent was evaporated toyield a brown gum. The product was isolated by reverse phase HPLC (0.1%TFA, water/acetonitrile gradient) to yield the product. MH⁺=354.

1.41-(5-Carboxypentyl)-2,3-dimethyl-3-(4-sulfobutyl)-4,5,6,7-tetrafluoro-3H-indolium

Tetra-fluorinated indole (from 1.3) (150 mg, 4.2×10⁻⁴ mol, 1 eq.) washeated at 140° C. with bromo-hexanoic acid (15 g, 0.073 mol, 260 eq) for24 hr under nitrogen. The product was triturated with diethyl ether anddried under vacuum to yield a brown mass. The major component wasconfirmed as1-(5-carboxypentyl)-2,3-dimethyl-3-(4-sulfobutyl)-4,5,6,7-tetrafluoro-3H-indolium[4] by LC-MS and was used without further purification. MH⁺=470.

1.52,3-Dimethyl-4,5,6,7-tetrafluoro-1,3-bis(4-sulphonatobutyl)-3H-indolium,disodium salt

Tetra-fluorinated indole from 1.3 (100 mg, 2.8×10⁻⁴ mol, 1 eq.) washeated at 140° C. with butane sultone (10 g, 0.073 mol) for 24 hr undernitrogen. The product was triturated with diethyl ether and dried undervacuum to yield a brown mass. The major component was confirmed as2,3-dimethyl-4,5,6,7-tetrafluoro-1,3-bis(4-sulphonatobutyl)-3H-indolium,disodium salt by LC-MS and was used without further purification.M⁺=490.

1.62-{(1E,3E,5E)-5-[1-(5-Carboxypentyl)-4,5,6,7-tetrafluoro-3-methyl-3-(4-sulfobutyl)-1,3-dihydro-2H-indol-2-ylidene]penta-1,3-dienyl}-4,5,6,7-tetrafluoro-3-methyl-1,3-bis(4-sulfobutyl)-3H-indolium(Cy5F) (Compound 1)

Alkylated indolenines (from 1.4 (5 mg) and 1.5 (1 eq.)) were dissolvedin ethanol and pyridine (250 μl). 1,1,1-Trimethoxypropene (1 eq.) wasadded and the solution stirred for two hours. Analysis by reverse phaseLC-MS observed three 650 nm absorbing products, nominally the twosymmetrical dyes and the desired product. The asymmetrical desiredproduct was isolated by HPLC and analysed by mass spectroscopy.Maldi-TOF MH⁺=993. Abs λ_(max)=644 nm (Aq). Em λ_(max)=659 nm. LC-MSindicates the main peak at 993 m/z for both isomers. HPLC-DAD gives >98%at 643 nm with peak purity>99%.

1.72-{(1E,3E,5E)-5-[1-{6-[(2,5-Dioxopyrrolidin-1-yl)oxy]-6-oxohexyl}-4,5,6,7-tetrafluoro-3-methyl-3-(4-sulfobutyl)-1,3-dihydro-2H-indol-2-ylidene]penta-1,3-dienyl}-4,5,6,7-tetrafluoro-3-methyl-1,3-bis(4-sulfobutyl)-3H-indolium(Cy5F NHS ester)

Cy5F free acid (500 μg) was dissolved in dimethyl-sulfoxide (500 μl)containing diisopropylethylamine (4% v/v) (20 μl). An aliquot of thissolution (1 μl) was retained as a control.Dipyrrolidino-(N-succinimidyloxy)carbenium hexafluorophosphate (1 mg)was added and the solution agitated for 1 hour prior to analysis by TLC(Merck RP-18_(F254), 1:1 water:acetonitrile+0.1% v/v TFA). Totalconversion of the starting material (Rf 0.4) to a new product spot (Rf0.36) was observed. The product (Cy5F NHS ester) was used immediatelyfor antibody conjugation without further purification.

1.8 Antibody Conjugation

Affinity purified Goat Anti-Mouse antibody IgG (100 μg) (Rockland Inc.Catalogue No. 610-101-121) was transferred to a pre-soakedSlide-A-Lyzer® dialysis cassette (Pierce Biotechnology; 10K MW cutoff)and dialysed (0.1 M NaHCO₃ buffer, pH 9.2). The dialysed product wasmixed with Cy5F NHS ester (from 1.7) (49 μl of a 1 mg/ml solution) andstirred at room temperature for 1 h. The reaction mixture wastransferred to a pre-soaked Slide-A-Lyzer® dialysis cassette (PierceBiotechnology; 10K MW cutoff) and dialyzed (0.1 M PBS-pH 7.2; 2×2 l) at4° C. for 48 h. The presence of dye-labeled antibody post dialysis wasdetermined by UV/Vis. spectroscopy, λ_(max) 645 nm, A=0.3 AU.

2.2-{(1E,3E,5E,7E)-7-[1-(5-Carboxypentyl)-4,5,6,7-tetrafluoro-3-methyl-3-(4-sulfobutyl)-1,3-dihydro-2H-indol-2-ylidene]hepta-1,3-5-trienyl}-4,5,6,7-tetrafluoro-3-methyl-1,3-bis(4-sulfobutyl)-3H-indolium(Cy7F) (Compound 2)

2.1 Alkylated indole (from 1.5) (0.1 mmol) andN-[5-(phenylamino)-2,4-pentadienylidene (Aldrich) (0.06 mmol) wereheated for 30 minutes at 120° C. in a 1:1 mixture of acetic acid andacetic anhydride (1.5 ml). This was cooled to ambient temperature andalkylated indole (from 1.4) was added (0.07 mmol) with potassium acetate(8 eq.). The mixture was stirred at 40° C. overnight, whereby a darkgreen solution was observed. The cooled solution was purified by HPLC(5-95% (Acetonitrile (0.1% TFA) gradient in water (0.1% TFA). Twoisomeric products were identified (R_(T)=7.98 m and 8.5 min) by LC-MSboth with m/z=1019 (M+1).

3.3-(5-Carboxypentyl)-3-methyl-5-sulfo-1-(4-sulfobutyl)-2-{1E,3E,5E}-5-[4,5,6,7-tetrafluoro-3-methyl-1,3-bis(4-sulfobutyl)-1,3-dihydro-2H-indol-2-ylidene]penta-1,3-dienyl}-3H-indolium(Compound 3)

3.1 3-(Carboxypentyl)-2,3-dimethylindolenine-5-sulfonic acid

4-Hydrazinobenzenesulfonic acid (13.42 g, 71.4 mmol) was added to7-methyl-8-oxononanoic acid (20 g, ˜107 mmol) in a 1 l round bottomedflask, together with acetic acid (71 ml). The pink suspension wasstirred at 140° C. for 5 hours. The reaction mixture was rotaryevaporated to yield a red/brown oil. The oil was triturated with diethylether (4×50 ml) to produce a semi-solid sticky mass. This was dissolvedin water (200 ml) and purified by flash chromatography. The product waseluted with 10% acetonitrile/water+0.1% TFA as a yellow band. HPLCanalysis showed the yellow band to be of adequate purity and separatedfrom the major impurity at 23.5 minutes. Yield 18 g.

3.2 2,3-Dimethyl-3-(5-carboxypentyl)-5-sulfo-1-(3-sulfobutyl)indolium,disodium salt

3-(Carboxypentyl)-2,3-dimethylindolenine-5-sulfonic acid (from 3.1) (5g, 14.5 mmol) and sodium acetate (1.55 g, 19.2 mmol) were dissolved inmethanol (˜30 ml) and the mixture was stirred for 5 minutes. The mixturewas then rotary evaporated three times from methanol to produce a yellowfluffy solid. Butane sultone (10 g, 5 eq) in sulfolan (50 ml) was addedand the mixture heated with stirring for 4 hours at 150° C. A red solidwas deposited on the bottom of the flask. A sample of the red solid wasanalysed by reverse phase (C18) HPLC (Jupiter 10μ C₁₈ 4.6 mm×250 mm)with 100% water (0.1% TFA) to 50% acetonitrile (0.1% TFA) over 30minutes and UV detection at 274 nm. The reaction mixture was allowed tocool and stored at +2° C. The reaction mixture was warmed and pouredinto stirring diethyl ether (500 ml), the ether was decanted off toleave a pink solid that was isolated.

3.32,3-Dimethyl-4,5,6,7-tetrafluoro-1,3-bis(4-sulphonatobutyl)-3H-indolium,disodium salt

Tetra-fluorinated indole (from 1.3) (100 mg, 2.8×10⁻⁴ mol, 1 eq.) washeated at 140° C. with butane sultone (10 g, 0.073 mol) for 24 hr undernitrogen. The product was triturated with diethyl ether and dried undervacuum to yield a brown mass. The major component was confirmed as (1.5)by LC-MS and was used without further purification. M⁺=491.

3.43-(5-Carboxypentyl)-3-methyl-5-sulfo-1-(4-sulfobutyl)-2-{1E,3E,5E}-5-[4,5,6,7-tetrafluoro-3-methyl-1,3-bis(4-sulfobutyl)-1,3-dihydro-2H-indol-2-ylidene]penta-1,3-dienyl}-3H-indolium(Compound 3)

2,3-Dimethyl-3-(5-carboxypentyl)-5-sulfo-1-(3-sulfobutyl)indolium,disodium salt (from 3.2) (3.4 g, 6.84 mmol) was dissolved in acetic acid(25 ml) containing triethylamine (2.5 ml). Malonaldehyde bisphenylimineHCl (4.15 g, 16 mmol) was added and the mixture heated at 140° C. withstirring overnight. The reaction mixture was rotary evaporated to yielda red oil which was dissolved in water (50 ml) and acetonitrile (30 ml).This was filtered and purified to yield 1 g of dye intermediate. 24 mg(3.9×10⁻⁵ mol, 4.9 molar equiv.) was dissolved in 1 ml solution ofacetic acid:acetic anhydride 1:1 v/v to form a bright red solution.2,3-Dimethyl-4,5,6,7-tetrafluoro-1,3-bis(4-sulphonatobutyl)-3H-indolium,disodium salt (4 mg, 8.12×10⁻⁶ mol) and potassium acetate (50 mg), wereadded and the reaction was stirred at 120° C. for 1 hour. The crudeblue/green material was purified by reverse phase preparative HPLC toyield an intense blue product. Rotary evaporation and freeze dryingresulted in 1 mg blue solid (12% yield). M⁺=1000.67.

4.1-(5-Carboxypentyl)-3-methyl-5-sulfo-3-(4-sulfobutyl)-2-{(1E,3E,5E)-5-[4,5,6,7-tetrafluoro-3-methyl-1,3-bis(4-sulfobutyl)-1,3-dihydro-2H-indol-2-ylidene]penta-1,3-dienyl}-3H-indolium(Compound 4)

4.1 Dye Intermediate

1-(Carboxypentynyl)-2,3,3-trimethyl-indoleninium-5-sulfonate (900 mg,1.91 mmol), malonaldehyde bisphenylimine (320 mg, 1.24 mmol, 0.9 eq),acetic anhydride (12 ml) and acetic acid (6 ml) were heated at 120° C.for 1 hour. The red/orange mixture was allowed to cool. UV and HPLCanalysis show the reaction to have gone to completion. The material wasused in the following stage (4.2) without further analysis.

4.21-(5-Carboxypentyl)-3-methyl-5-sulfo-3-(4-sulfobutyl)-2-{(1E,3E,5E)-5-[4,5,6,7-tetrafluoro-3-methyl-1,3-bis(4-sulfobutyl)-1,3-dihydro-2H-indol-2-ylidene]penta-1,3-dienyl}-3H-indolium(Compound 4)

2,3-Dimethyl-3-(5-carboxypentyl)-5-sulfo-1-(3-sulfobutyl)indolium,disodium salt (5 mg) was added to the dye intermediate from 5.1 (50mg/ml, 100 μl) and potassium acetate (8 mg, 8 eq.). The reaction wasstirred at room temperature overnight to yield a deep blue solution. Theproduct was isolated by reverse phase HPLC (acetonitrile/water/0.1%trifluoroacetic acid). M⁺=879. UV/Vis λ_(max) 647 nm. Yield 4.5 mg.

5.2-{(1E,3E,5E)-5-[1-(5-Carboxypentyl)-4,5,6,7-tetrafluoro-3-methyl-3-(4-sulfobutyl)-1,3-dihydro-2H-indol-2-ylidene]penta-1,3-dienyl}-4,6-difluoro-3-methyl-1,3-bis(4-sulfobutyl)-3H-indolium(Compound 5)

5.1 4,6-Difluoro-2,3-dimethyl-3-(4-sulfobutyl)-3H-indole

To 3,5-difluorophenyl hydrazine hydrochloride (1 g) in acetic acid (20ml) was added 5-methyl-6-oxoheptane-1-sulphonic acid (1.6 g) and thesolution heated to reflux overnight. The volatiles were removed on arotary evaporator to give the crude product, 50 mg of which was purifiedby preparative HPLC. The relevant fractions were combined, concentratedon a rotary evaporator and freeze dried to give the desired product (19mg). M⁺=317

5.2 4,6-Difluoro-2,3-dimethyl-1,3-bis(4-sulfobutyl)-3H-indolium

To 4,6-difluoro-2,3-dimethyl-3-(4-sulfobutyl)-3H-indole (1 g of nonpurified material) was added butane sultone (10 ml) and the solutionheated to 140° C. for 2 days. On cooling, the product was extracted intowater and purified by preparative HPLC. The relevant fractions werecombined, concentrated on a rotary evaporator and freeze dried to givethe desired product (90 mg). M⁺=454

5.36-[(2E)-4,5,6,7-Tetrafluoro-3-methyl-2-[(2E,4E)-4-(phenylimino)but-2-enylidene]-3-(4-sulfobutyl)-2,3-dihydro-1H-indol-1-yl]hexanoicacid

To1-(5-carboxypentyl)-4,5,6,7-tetrafluoro-3-methyl-3-(4-sulfobutyl)-3H-indolium(12 mg) was added malonaldehyde bis(phenylimine) monohydrochloride (7mg), acetic anhydride (2 ml) and acetic acid (1 ml). This mixture washeated to 120° C. for 1 hour 50 minutes after which time it was cooledto room temperature. This crude reaction mixture (ca. 5 mg/ml product)was used without purification as further described.

5.42-{(1E,3E,5E)-5-[1-(5-Carboxypentyl)-4,5,6,7-tetrafluoro-3-methyl-3-(4-sulfobutyl)-1,3-dihydro-2H-indol-2-ylidene]penta-1,3-dienyl}-4,6-difluoro-3-methyl-1,3-bis(4-sulfobutyl)-3H-indolium(Compound 5)

To6-[(2E)-4,5,6,7-tetrafluoro-3-methyl-2-[(2E,4E)-4-(phenylimino)but-2-enylidene]-3-(4-sulfobutyl)-2,3-dihydro-1H-indol-1-yl]hexanoicacid (0.4 ml of ca. 5 mg/ml) was added4,6-difluoro-2,3-dimethyl-3-(4-sulfobutyl)-3H-indole (2 mg) andpotassium acetate (4.4 mg). On agitation overnight, preparative HPLC wasperformed, the relevant fractions were combined, concentrated on arotary evaporator and freeze dried to give the desired product (4 mg).M⁺=957

6.2-{(1E,3E,5E)-5-[1-(5-Carboxypentyl)-4,6-difluoro-3,3-dimethyl-1,3-dihydro-2H-indol-2-ylidene]penta-1,3-dienyl}-4,5,6,7-tetrafluoro-3-methyl-1,3-bis(4-sulfobutyl)-3H-indolium(compound 6)

6.1 4,6-Difluoro-2,3,3-trimethyl-3H-indole

To 3,5-difluorophenyl hydrazine hydrochloride (2.6 g) in ethanol (30 ml)was added 3-methyl-2-butanone (6 ml) and the solution heated to 78° C.for 3 hours. After this time the mixture was cooled and the volatilesremoved on a rotary evaporator to give a red solid. To this was addedpolyphosphoric acid (10 g) and acetic acid (25 ml). This mixture washeated to 120° C. for 3 hours and then left to cool overnight. Thevolatiles were removed on a rotary evaporator and the pH adjusted topH5.5 by the addition of sodium hydroxide to give the crude productwhich was purified by flash chromatography (silica gel/hexane/ethylacetate). The relevant fractions were combined and concentrated on arotary evaporator to give the desired product (2 g). MH⁺=196

6.2 1-(5-Carboxypentyl)-4,6-difluoro-2,3,3-trimethyl-3H-indolium

To 4,6-difluoro-2,3,3-trimethyl-3H-indole (2 g) was added6-bromohexanoic acid (5 g) and sulfolan (5 ml) and the mixture heated to140° C. for 5 hours. On cooling the mixture was diluted with water andpreparative HPLC performed to give the desired product (1.4 g). M⁺=310

6.36-[(2E)-4,6-Difluoro-3,3-dimethyl-2-[(2E,4E)-4-(phenylimino)but-2-enylidene]-2,3-dihydro-1H-indol-1-yl]hexanoicacid

To 1-(5-carboxypentyl)-4,6-difluoro-2,3,3-trimethyl-3H-indolium (50 mg)was added malonaldehyde bis(phenylimine) monohydrochloride (37.6 mg),acetic anhydride (2 ml) and acetic acid (1 ml). This mixture was heatedto 120° C. for 1 hour after which time it was cooled to roomtemperature. This crude reaction mixture (ca. 32 mg/ml product) was usedwithout purification as further described.

6.42-{(1E,3E,5E)-5-[1-(5-Carboxypentyl)-4,6-difluoro-3,3-dimethyl-1,3-dihydro-2H-indol-2-ylidene]penta-1,3-dienyl}-4,5,6,7-tetrafluoro-3-methyl-1,3-bis(4-sulfobutyl)-3H-indolium(Compound 6)

To6-[(2E)-4,6-difluoro-3,3-dimethyl-2-[(2E,4E)-4-(phenylimino)but-2-enylidene]-2,3-dihydro-1H-indol-1-yl]hexanoicacid (0.04 mmol) was added4,5,6,7-tetrafluoro-2,3-dimethyl-1,3-bis(4-sulfobutyl)-3H-indolium (19.6mg) and potassium acetate (35 mg). On agitation overnight, preparativeHPLC was performed, the relevant fractions were combined, concentratedon a rotary evaporator and freeze dried to give the desired product (6mg). M⁺=835

6.52-[(1E,3E,5E)-5-(1-{6-[(2,5-Dioxopyrrolidin-1-yl)oxy]-6-oxohexyl}-4,6-difluoro-3,3-dimethyl-1,3-dihydro-2H-indol-2-ylidene)penta-1,3-dienyl]-4,5,6,7-tetrafluoro-3-methyl-1,3-bis(4-sulfobutyl)-3H-indolium

To2-{(1E,3E,5E)-5-[1-(5-carboxypentyl)-4,6-difluoro-3,3-dimethyl-1,3-dihydro-2H-indol-2-ylidene]penta-1,3-dienyl}-4,5,6,7-tetrafluoro-3-methyl-1,3-bis(4-sulfobutyl)-3H-indolium(2 mg) was added DMSO (1 ml), dipyrrolidino-(N-succinimidyloxy)carbeniumhexafluorophosphate (4 mg) and diisopropyl ethylamine (40 ul). Afteragitation for 3 hours, the reaction mixture subjected to preparativeHPLC to give the desired product (0.4 mg). M⁺=932

7.1-(5-Carboxypentyl)-2-{(1E,3E,5E)-5-[1-(5-carboxypentyl)-4,6-difluoro-3,3-dimethyl-1,3-dihydro-2H-indol-2-ylidene]penta-1,3-dienyl}-4,5,6,7-tetrafluoro-3-methyl-3-(4-sulfobutyl)-3H-indolium(Compound 7)

7.11-(5-Carboxypentyl)-4,6-difluoro-2,3-dimethyl-3-(4-sulfobutyl)-3H-indolium

To 4,6-difluoro-2,3-dimethyl-3-(4-sulfobutyl)-3H-indole (0.8 g of nonpurified material) was added 6-bromohexanoic acid (1.6 g) and thesolution heated to 140° C. for 2 days. On cooling, the product wasdiluted with acetonitrile and purified by preparative HPLC in two shots.The relevant fractions were combined, concentrated on a rotaryevaporator and freeze dried to give the desired product (110 mg). M⁺=432

7.21-(5-Carboxypentyl)-2-{(1E,3E,5E)-5-[1-(5-carboxypentyl)-4,6-difluoro-3,3-dimethyl-1,3-dihydro-2H-indol-2-ylidene]penta-1,3-dienyl}-4,5,6,7-tetrafluoro-3-methyl-3-(4-sulfobutyl)-3H-indolium(Compound 7)

To6-[(2E)-4,5,6,7-tetrafluoro-3-methyl-2-[(2E,4E)-4-(phenylimino)but-2-enylidene]-3-(4-sulfobutyl)-2,3-dihydro-1H-indol-1-yl]hexanoicacid (0.7 ml of ca. 5 mg/ml) was added1-(5-carboxypentyl)-4,6-difluoro-2,3-dimethyl-3-(4-sulfobutyl)-3H-indolium(2.8 mg) and potassium acetate (6 mg). On agitation overnight,preparative HPLC was performed, the relevant fractions were combined,concentrated on a rotary evaporator and freeze dried to give the desiredproduct (1.6 mg). M⁺=936

8.2-{(1E,3E,5E)-5-[1-(5-Carboxypentyl)-4,5,6,7-tetrafluoro-3-methyl-3-(4-sulfobutyl)-1,3-dihydro-2H-indol-2-ylidene]penta-1,3-dienyl}-3-methyl-1,3-bis(4-sulfobutyl)-5-[(trifluoromethyl)sulfonyl]-3H-indolium(Compound 8)

8.1 4-(Trifluoromethylthio)phenylhydrazine

To 4-(trifluoromethylthio)aniline (5 g) was added cHCl (300 ml). Atbetween 0° C. and −5° C. was added sodium nitrite (2.5 g) in water (50ml) over 15 min. After a further 25 min at this temperature the mixturewas filtered and tin (II) chloride dihydrate added to the solution over5 min. The mixture was allowed to warm to room temperature with stirringovernight. The resulting precipitate was filtered to give a white solid(3.3 g). M⁺=208

8.2 5-(Trifluoromethylthio)-2,3-dimethyl-3-(4-sulfobutyl)-3H-indole

To 4-(trifluoromethylthio)phenylhydrazine (1 g) in acetic acid (40 ml)was added 5-methyl-6-oxoheptane-1-sulphonic acid (1.4 g) and thesolution heated to reflux overnight. The volatiles were removed on arotary evaporator to give the crude product, 1.1 g of which was purifiedby preparative HPLC. The relevant fractions were combined, concentratedon a rotary evaporator and freeze dried to give the desired product (260mg). M⁺=381

8.35-(Trifluoromethylthio)-2,3-dimethyl-1,3-bis(4-sulfobutyl)-3H-indolium

To 5-(trifluoromethylthio)-2,3-dimethyl-3-(4-sulfobutyl)-3H-indole (36mg) was added butane sultone (2 ml) and the solution heated to 140° C.overnight. On cooling, the product was extracted into water and purifiedby preparative HPLC. The relevant fractions were combined, concentratedon a rotary evaporator and freeze dried to give the desired product (15mg). M⁺=518

8.45-(Trifluoromethylsulfonyl)-2,3-dimethyl-1,3-bis(4-sulfobutyl)-3H-indolium

To5-(trifluoromethylthio)-2,3-dimethyl-1,3-bis(4-sulphonatobutyl)-3H-indolium(4 mg) was added concentrated sulphuric acid (0.4 ml) and chromiumtrioxide (5 mg). After 60 min the mixture was diluted with water andpurified by preparative HPLC. The relevant fractions were combined,concentrated on a rotary evaporator and freeze dried to give the desiredproduct (1 mg). M⁺=550

8.52-{(1E,3E,5E)-5-[1-(5-Carboxypentyl)-4,5,6,7-tetrafluoro-3-methyl-3-(4-sulfobutyl)-1,3-dihydro-2H-indol-2-ylidene]penta-1,3-dienyl}-3-methyl-1,3-bis(4-sulfobutyl)-5-[(trifluoromethyl)sulfonyl]-3H-indolium(Compound 8)

To6-[(2E)-4,5,6,7-tetrafluoro-3-methyl-2-[(2E,4E)-4-(phenylimino)but-2-enylidene]-3-(4-sulfobutyl)-2,3-dihydro-1H-indol-1-yl]hexanoicacid (0.21 ml of ca. 5 mg/ml) was added5-(trifluoromethylsulfonyl)-2,3-dimethyl-1,3-bis(4-sulphonatobutyl)-3H-indolium(1 mg) and potassium acetate (4 mg). On agitation overnight, preparativeHPLC was performed, the relevant fractions were combined, concentratedon a rotary evaporator and freeze dried to give the desired product (0.1mg). M⁺=1054

9.2-{(1E,3E,5E)-5-[1-(4-Sulfobutyl)-4,6-bis(trifluoromethyl)-3-methyl-3-(5-carboxypentyl))-1,3-dihydro-2H-indol-2-ylidene]penta-1,3-dienyl}-4,6-difluoro-3-methyl-1,3-bis(4-sulfobutyl)-3H-indolium(compound 9)

9.1 4,6-Bis(trifluoromethyl)-2,3-dimethyl-3-(5-carboxypentyl)-3H-indole

To 3,5-bis(trifluoromethyl)phenyl hydrazine hydrochloride(1 g) in aceticacid (20 ml) was added 7-methyl-8-oxononanoic acid (0.66 g) and thesolution heated to reflux for 5 hours. The volatiles were removed on arotary evaporator to give the crude product, 1 ml of which was purifiedby preparative HPLC. The relevant fractions were combined, concentratedon a rotary evaporator and freeze dried to give the desired product (17mg). M⁺=395

9.23-(5-Carboxypentyl)-4,6-bis(trifluoromethyl)-2,3-dimethyl-1-(4-sulfobutyl)-3H-indolium

To 4,6-bis(trifluoromethyl)-2,3-dimethyl-3-(5-carboxypentyl)-3H-indole(300 mg of non purified material) was added butane sultone (3 ml) andthe solution heated to 140° C. for 1 day. On cooling, the product wasextracted into water and purified by preparative HPLC. The relevantfractions were combined, concentrated on a rotary evaporator and freezedried to give the desired product (19 mg). M⁺=532

9.32-{(1E,3E,5E)-5-[1-(4-Sulfobutyl)-4,6-Bis(trifluoromethyl)-3-methyl-3-(5-carboxypentyl))-1,3-dihydro-2H-indol-2-ylidene]penta-1,3-dienyl}-4,6-difluoro-3-methyl-1,3-bis(4-sulfobutyl)-3H-indolium(Compound 9)

To3-(5-carboxypentyl)-4,6-bis(trifluoromethyl)-2,3-dimethyl-1-(4-sulfobutyl)-3H-indolium(3.7 mg) was added malonaldehyde bis(phenylimine) monohydrochloride (1.6mg), acetic anhydride (0.5 ml) and acetic acid (0.25 ml). This mixturewas heated at 120° C. for 1 hour and then cooled to room temperature.4,6-Difluoro-2,3-dimethyl-1,3-bis(4-sulfobutyl)-3H-indolium (3.15 mg)and potassium acetate (6.1 mg) was added. On agitation overnight,preparative HPLC was performed, the relevant fractions were combined,concentrated on a rotary evaporator and freeze dried to give the desiredproduct (0.5 mg). M⁺=1021

10.2-{(1E,3E,5E)-5-[1-(5-Carboxypentyl)-4,5,6,7-tetrafluoro-3-methyl-3-(4-sulfobutyl)-1,3-dihydro-2H-indol-2-ylidene]penta-1,3-dienyl}-3-methyl-1,3-bis(4-sulfobutyl)-6-(trifluoromethyl)-3H-indolium(Compound 10)

10.1 6-Trifluoromethyl-2,3-dimethyl-3-(4-sulfobutyl)-3H-indole

To 3-(trifluoromethyl)phenyl hydrazine (1 g) in acetic acid (30 ml) wasadded 5-methyl-6-oxoheptane-1-sulphonic acid (1.3 g) and the solutionheated to reflux overnight. The volatiles were removed on a rotaryevaporator to give the crude product, 1.3 g of which was purified bypreparative HPLC. The relevant fractions were combined, concentrated ona rotary evaporator and freeze dried to give the desired product (55mg). M⁺=349

10.2 6-Trifluoromethyl-2,3-dimethyl-1,3-bis(4-sulfobutyl)-3H-indolium

To 6-trifluoromethyl-2,3-dimethyl-3-(4-sulfobutyl)-3H-indole (27 mg) wasadded butane sultone (2 ml) and the solution heated to 140° C. for 3days. On cooling, the product was extracted into water and purified bypreparative HPLC. The relevant fractions were combined, concentrated ona rotary evaporator and freeze dried to give the desired product (14mg). M⁺=486

10.32-{(1E,3E,5E)-5-[1-(5-Carboxypentyl)-4,5,6,7-tetrafluoro-3-methyl-3-(4-sulfobutyl)-1,3-dihydro-2H-indol-2-ylidene]penta-1,3-dienyl}-3-methyl-1,3-bis(4-sulfobutyl)-6-(trifluoromethyl)-3H-indolium(Compound 10)

To6-[(2E)-4,5,6,7-tetrafluoro-3-methyl-2-[(2E,4E)-4-(phenylimino)but-2-enylidene]-3-(4-sulfobutyl)-2,3-dihydro-1H-indol-1-yl]hexanoicacid (0.65 ml of ca. 5 mg/ml) was added6-trifluoromethyl-2,3-dimethyl-1,3-bis(4-sulphonatobutyl)-3H-indolium(2.6 mg) and potassium acetate (6 mg). On agitation overnight,preparative HPLC was performed, the relevant fractions were combined,concentrated on a rotary evaporator and freeze dried to give the desiredproduct (0.1 mg). M⁺=989

11.2-{(1E,3E,5E)-5-[1-(5-Carboxypentyl)-4,5,6,7-tetrafluoro-3-methyl-3-(4-sulfobutyl)-1,3-dihydro-2H-indol-2-ylidene]penta-1,3-dienyl}-4,5,6,7,8,9-hexafluoro-1,1-dimethyl-3-(4-sulfobutyl)-1H-benzo[e]indolium(Compound 11)

11.1 (1,2,3,4,6,7,9-Heptafluoro-5H-benzo[7]annulen-8-yl)hydrazine

To octafluoronaphthalene (1.2 g) was added ethanol (20 ml) and hydrazinehydrate (0.6 ml). The mixture was heated to 50° C. overnight. On coolingthe mixture was poured into water and the resulting precipitate filteredoff. Air drying gave 1 g of buff material which was used without furtherpurification.

M⁺=284

11.23-Methylbutan-2-one(1,2,3,4,6,7,9-heptafluoro-5H-benzo[7]annulen-8-yl)hydrazone

To (1,2,3,4,6,7,9-heptafluoro-5H-benzo[7]annulen-8-yl)hydrazine (1 g)was added ethanol (20 ml) and 3-methyl-2-butanone (0.38 g). The mixturewas heated to reflux for 3 hours after which time the volatiles wereremoved on a rotary evaporator to give a solid (1 g) which was used inthe next step. M⁺=352

11.3 4,5,6,7,8,9-Hexafluoro-1,1,2-trimethyl-1H-benzo[e]indole

To3-methylbutan-2-one(1,2,3,4,6,7,9-heptafluoro-5H-benzo[7]annulen-8-yl)hydrazone(44 mg) was added tetralin (2 ml) and the mixture heated to 200° C.overnight. On cooling to room temperature the mixture was subjected topreparative HPLC. The relevant fractions were combined, concentrated ona rotary evaporator and freeze dried to give the desired product (2 mg).M⁺=317

11.44,5,6,7,8,9-Hexafluoro-1,1,2-trimethyl-3-(4-sulfobutyl)-1H-benzo[e]indolium

To 4,5,6,7,8,9-hexafluoro-1,1,2-trimethyl-1H-benzo[e]indole (2 mg) wasadded butane sultone (200 ul) and the mixture heated to 140° C.overnight. After preparative HPLC, the relevant fractions were combined,concentrated on a rotary evaporator and freeze dried to give the desiredproduct (1 mg). M⁺=454

11.52-{(1E,3E,5E)-5-[1-(5-Carboxypentyl)-4,5,6,7-tetrafluoro-3-methyl-3-(4-sulfobutyl)-1,3-dihydro-2H-indol-2-ylidene]penta-1,3-dienyl}-4,5,6,7,8,9-hexafluoro-1,1-dimethyl-3-(4-sulfobutyl)-1H-benzo[e]indolium(compound 11)

To6-[(2E)-4,5,6,7-tetrafluoro-3-methyl-2-[(2E,4E)-4-(phenylimino)but-2-enylidene]-3-(4-sulfobutyl)-2,3-dihydro-1H-indol-1-yl]hexanoicacid (0.18 ml of ca. 5 mg/ml) was added4,5,6,7,8,9-Hexafluoro-1,1,2-trimethyl-3-(4-sulfobutyl)-1H-benzo[e]indolium(0.7 mg) and potassium acetate (4 mg). On agitation overnight,preparative HPLC was performed, the relevant fractions were combined,concentrated on a rotary evaporator and freeze dried to give the desiredproduct (0.1 mg). M⁺=957

12.2-{(1E,3E,5E)-5-[1-(5-Carboxypentyl)-4,6-difluoro-3,3-dimethyl-1,3-dihydro-2H-indol-2-ylidene]penta-1,3-dienyl}-6-fluoro-3-methyl-1,3-bis(4-sulfobutyl)-4-(trifluoromethyl)-3H-indolium(Compound 12)

12.1 4-Trifluoromethyl-6-fluoro-2,3-dimethyl-3-(4-sulfobutyl)-3H-indole

To 3-trifluoromethyl-5-fluorophenyl hydrazine (2 g) was added5-methyl-6-oxoheptane-1-sulfonic acid (3 g) and acetic acid (60 ml) andthe mixture heated to 140° C. for 5 hours. After this time the volatileswere removed on a rotary evaporator and the residue dissolved in water(10 ml), filtered and purified by preparative HPLC to give the desiredproduct (790 mg). MH⁺=368

12.24-Trifluoromethyl-6-fluoro-2,3-dimethyl-1,3-bis(4-sulfobutyl)-3H-indolium

To 4-trifluoromethyl-6-fluoro-2,3-dimethyl-3-(4-sulfobutyl)-3H-indole(790 mg) was added butane sultone (10 ml) and the mixture heated at 140°C. overnight. The mixture was diluted with water (4 ml), filtered andpurified by preparative HPLC to give the desired material (1 g). MH⁺=505

12.32-{(1E,3E,5E)-5-[1-(5-Carboxypentyl)-4,6-difluoro-3,3-dimethyl-1,3-dihydro-2H-indol-2-ylidene]penta-1,3-dienyl}-6-fluoro-3-methyl-1,3-bis(4-sulfobutyl)-4-(trifluoromethyl)-3H-indolium(Compound 12)

To6-[(2E)-4,6-difluoro-3,3-dimethyl-2-[(2E,4E)-4-(phenylimino)but-2-enylidene]-2,3-dihydro-1H-indol-1-yl]hexanoicacid (0.04 mmol) was added4-trifluoromethyl-6-fluoro-2,3-dimethyl-1,3-bis(4-sulfobutyl)-3H-indolium(20 mg) and potassium acetate (35 mg). On agitation overnight,preparative HPLC was performed, the relevant fractions were combined,concentrated on a rotary evaporator and freeze dried to give the desiredproduct (4.2 mg). M⁺=849

13.2-{(1E,3E,5E)-5-[1-(5-Carboxypentyl)-4,6-difluoro-3,3-dimethyl-1,3-dihydro-2H-indol-2-ylidene]penta-1,3-dienyl}-4,6-difluoro-3-methyl-1,3-bis(4-sulfobutyl)-3H-indolium(Compound 13)

13.1 To6-[(2E)-4,6-difluoro-3,3-dimethyl-2-[(2E,4E)-4-(phenylimino)but-2-enylidene]-2,3-dihydro-1H-indol-1-yl]hexanoicacid (0.04 mmol) was added4,6-difluoro-2,3-dimethyl-1,3-bis(4-sulfobutyl)-3H-indolium (18.1 mg)and potassium acetate (35 mg). On agitation overnight, preparative HPLCwas performed, the relevant fractions were combined, concentrated on arotary evaporator and freeze dried to give the desired product (10.7mg). MH⁺=800

14.2-{(1E,3E,5E)-5-[1-(5-Carboxypentyl)-4,6-difluoro-3,3-dimethyl-1,3-dihydro-2H-indol-2-ylidene]penta-1,3-dienyl}-3-methyl-5-sulfo-1,3-bis(4-sulfobutyl)-3H-indolium(Compound 14)

14.1 2,3-Dimethyl-3-(4-sulfobutyl)-5-sulfo-3H-indole

To hydrazinobenzene sulfonic acid (40 g) was added acetic acid (500 ml)and 5-methyl-6-oxoheptane-1-sulfonic acid (60 g). The mixture was heatedto 150° C. for 6 hours and then cooled to room temperature. Afterfiltration the volatiles were removed on a rotary evaporator to give asolid (110 g), 100 g of which was dissolved in methanol (1 L) andpotassium hydroxide (38 g) in methanol (300 ml) added. The mixture wasstirred for 1 hour and then stood overnight. The liquid layer wasdecanted off and the volatiles removed on a rotary evaporator to givethe product as a pale yellow solid (80 g).

14.2 2,3-Dimethyl-1,3-bis(4-sulfobutyl)-5-sulfo-3H-indole

To 2,3-dimethyl-3-(4-sulfobutyl)-5-sulfo-3H-indole (10 g) was addedbutane sultone (30 g) and the mixture heated to 140° C. overnight. Oncooling to room temperature, sulfolan (20 ml) was added and the mixturepoured into diethyl ether (500 ml). The resultant precipitate wasremoved by filtration and dried to give the desired product (12 g).

14.32-{(1E,3E,5E)-5-[1-(5-Carboxypentyl)-4,6-difluoro-3,3-dimethyl-1,3-dihydro-2H-indol-2-ylidene]penta-1,3-dienyl}-3-methyl-5-sulfo-1,3-bis(4-sulfobutyl)-3H-indolium(Compound 14)

To6-[(2E)-4,6-difluoro-3,3-dimethyl-2-[(2E,4E)-4-(phenylimino)but-2-enylidene]-2,3-dihydro-1H-indol-1-yl]hexanoicacid (0.04 mmol) was added2,3-dimethyl-1,3-bis(4-sulfobutyl)-5-sulfo-3H-indole (20 mg) andpotassium acetate (35 mg). On agitation overnight, preparative HPLC wasperformed, the relevant fractions were combined, concentrated on arotary evaporator and freeze dried to give the desired product (8.9 mg).M⁺=843

15.2-{(1E,3E,5E)-5-[3-(5-Carboxypentyl)-4,5,6,7-tetrafluoro-3-methyl-1-(4-sulfobutyl)-1,3-dihydro-2H-indol-2-ylidene]penta-1,3-dienyl}-4,5,6,7-tetrafluoro-3-methyl-1,3-bis-(4-sulfobutyl)-3H-indolium(Compound 15)

15.1 6-(4,5,6,7-Tetrafluoro-2,3-dimethyl-3H-indol-3-yl)hexanoic acid

To tetrafluorophenyl hydrazine (2 g) was added5-methyl-6-oxoheptane-1-carboxylic acid (3 g) and acetic acid (50 ml)and the mixture heated to 140° C. for 5 hours. On cooling the volatilecomponents were removed on a rotary evaporator and the residue dissolvedin water (10 ml), filtered and purified by preparative HPLC in 2 shotsto give the desired product (600 mg). MH⁺=332

15.23-(5-Carboxypentyl)-4,5,6,7-tetrafluoro-2,3-dimethyl-1-(4-sulfobutyl)-3H-indolium

To 6-(4,5,6,7-tetrafluoro-2,3-dimethyl-3H-indol-3-yl)hexanoic acid (600mg) was added butane sultone (4 ml) and the mixture heated to 140° C.overnight. On cooling the mixture was diluted with water (4 ml),filtered and purified by preparative HPLC to give the desired product(800 mg). MH⁺=468

15.32-{(1E,3E,5E)-5-[3-(5-Carboxypentyl)-4,5,6,7-tetrafluoro-3-methyl-1-(4-sulfobutyl)-1,3-dihydro-2H-indol-2-ylidene]penta-1,3-dienyl}-4,5,6,7-tetrafluoro-3-methyl-1,3-bis-(4-sulfobutyl)-3H-indolium(Compound 15)

To3-(5-carboxypentyl)-4,5,6,7-tetrafluoro-2,3-dimethyl-1-(4-sulfobutyl)-3H-indolium(19 mg) was added malonaldehyde bisphenylimine (9.4 mg), aceticanhydride (0.5 ml) and acetic acid (0.25 ml). The mixture was heated at120° C. for 1 hour and then allowed to cool to room temperature.2,3-Dimethyl-4,5,6,7-tetrafluoro-1,3-bis(4-sulphonatobutyl)-3H-indolium(20 mg) and potassium acetate (35.7 mg) were added and the mixturestirred overnight. Preparative HPLC purification gave the desiredproduct (4.5 mg). MH⁺=994

15.42-{(1E,3E,5E)-5-[3-{6-[(2,5-Dioxopyrrolidin-1-yl)oxy]-6-oxohexyl}-4,5,6,7-tetrafluoro-3-methyl-1-(4-sulfobutyl)-1,3-dihydro-2H-indol-2-ylidene]penta-1,3-dienyl}-4,5,6,7-tetrafluoro-3-methyl-1,3-bis(4-sulfobutyl)-3H-indolium

To2-{(1E,3E,5E)-5-[3-(5-carboxypentyl)-4,5,6,7-tetrafluoro-3-methyl-1-(4-sulfobutyl)-1,3-dihydro-2H-indol-2-ylidene]penta-1,3-dienyl}-4,5,6,7-tetrafluoro-3-methyl-1,3-bis-(4-sulfobutyl)-3H-indolium(2 mg) was added DMSO (1 ml), dipyrrolidino-(N-succinimidyloxy)carbeniumhexafluorophosphate (4 mg) and diisopropyl ethylamine (40 ul). Afteragitation for 3 hours, the reaction mixture was subjected to preparativeHPLC to give the desired product (0.7 mg). M⁺=1091

16.2-{(1E,3E,5E)-5-[3-(5-Carboxypentyl)-6-fluoro-3-methyl-1-(4-sulfobutyl)-4-(trifluoromethyl)-1,3-dihydro-2H-indol-2-ylidene]penta-1,3-dienyl}-4,5,6,7-tetrafluoro-3-methyl-1,3-bis(4-sulfobutyl)-3H-indolium(Compound 16)

16.1 6-(4-Trifluoromethyl-6-fluoro-2,3-dimethyl-3H-indol-3-yl)hexanoicacid

To 3-trifluoromethyl-5-fluorophenyl hydrazine (2 g) was added5-methyl-6-oxoheptane-1-carboxylic acid (3 g) and acetic acid (50 ml)and the mixture heated to 140° C. for 5 hours. On cooling the volatilecomponents were removed on a rotary evaporator and the residue dissolvedin water (10 ml), filtered and purified by preparative HPLC in 2 shotsto give the desired product (600 mg). MH⁺=346

16.23-(5-Carboxypentyl)-4-trifluoromethyl-6-fluoro-2,3-dimethyl-1-(4-sulfobutyl)-3H-indolium

To 6-(4-trifluoromethyl-6-fluoro-2,3-dimethyl-3H-indol-3-yl)hexanoicacid (600 mg) was added butane sultone (4 ml) and the mixture heated to140° C. overnight. On cooling the mixture was diluted with water (4 ml),filtered and purified by preparative HPLC to give the desired product(800 mg). MH⁺=483

16.32-{(1E,3E,5E)-5-[3-(5-carboxypentyl)-6-fluoro-3-methyl-1-(4-sulfobutyl)-4-(trifluoromethyl)-1,3-dihydro-2H-indol-2-ylidene]penta-1,3-dienyl}-4,5,6,7-tetrafluoro-3-methyl-1,3-bis(4-sulfobutyl)-3H-indolium(Compound 16)

To3-(5-carboxypentyl)-4-trifluoromethyl-6-fluoro-2,3-dimethyl-1-(4-sulfobutyl)-3H-indolium(50 mg) was added malonaldehyde bisphenylimine (24 mg), acetic anhydride(0.5 ml) and acetic acid (0.25 ml). The mixture was heated at 120° C.for 1 hour and then allowed to cool to room temperature.2,3-Dimethyl-4,5,6,7-tetrafluoro-1,3-bis(4-sulphonatobutyl)-3H-indolium(51 mg) and potassium acetate (92 mg) were added and the mixture stirredovernight. Preparative HPLC purification gave the desired product (19.5mg). MH⁺=1008

16.42-{(1E,3E,5E)-5-[3-{6-[(2,5-Dioxopyrrolidin-1-yl)oxy]-6-oxohexyl}-6-fluoro-3-methyl-1-(4-sulfobutyl)-4-(trifluoromethyl)-1,3-dihydro-2H-indol-2-ylidene]penta-1,3-dienyl}-4,5,6,7-tetrafluoro-3-methyl-1,3-bis(4-sulfobutyl)-3H-indolium

To2-{(1E,3E,5E)-5-[3-(5-carboxypentyl)-4-trifluoromethyl-6-fluoro-3-methyl-1,3-dihydro-2H-indol-2-ylidene]penta-1,3-dienyl}-4,5,6,7-tetrafluoro-3-methyl-1,3-bis(4-sulfobutyl)-3H-indolium(5 mg) was added DMSO (1 ml), dipyrrolidino-(N-succinimidyloxy)carbeniumhexafluorophosphate (10 mg) and diisopropyl ethylamine (40 ul). Afteragitation for 3 hours, the reaction mixture subjected to preparativeHPLC to give the desired product (2.1 mg). M⁺=1104

16.52-{(1E,3E,5E)-5-[3-(6-{[2-(2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl]amino}-6-oxohexyl)-4,5,6,7-tetrafluoro-3-methyl-1-(4-sulfobutyl)-1,3-dihydro-2H-indol-2-ylidene]penta-1,3-dienyl}-4,5,6,7-tetrafluoro-3-methyl-1,3-bis(4-sulfobutyl)-3H-indolium

To N-[2-(N-t-butoxycarbonyl)aminoethyl]maleimide (4 mg) was stirred with4M HCl in dioxin (100 ul) for 1 hour. After rotary evaporation,2-{(1E,3E,5E)-5-[3-{6-[(2,5-dioxopyrrolidin-1-yl)oxy]-6-oxohexyl}-6-fluoro-3-methyl-1-(4-sulfobutyl)-4-(trifluoromethyl)-1,3-dihydro-2H-indol-2-ylidene]penta-1,3-dienyl}-4,5,6,7-tetrafluoro-3-methyl-1,3-bis(4-sulfobutyl)-3H-indolium(2 mg) in DMF (1 ml) and diisopropyl ethylamine (40 ul) were added andthe mixture stirred for 2 hours. Preparative HPLC gave the desiredproduct (2.1 mg). M⁺=1129

17.2-{(1E,3E,5E)-5-[3-(5-Carboxypentyl)-4-trifluoromethyl-6-fluoro-3-methyl-1,3-dihydro-2H-indol-2-ylidene]penta-1,3-dienyl}-3-(5-carboxypentyl)-4-trifluoromethyl-6-fluoro-3-methyl-3H-indolium(Compound 17)

17.12-{(1E,3E,5E)-5-[3-(5-Carboxypentyl)-4-trifluoromethyl-6-fluoro-3-methyl-1,3-dihydro-2H-indol-2-ylidene]penta-1,3-dienyl}-3-(5-carboxypentyl)-4-trifluoromethyl-6-fluoro-3-methyl-3H-indolium(compound 17)

The experiment described in 16.3 also gave this material isolated viapreparative HPLC (8.5 mg). MH⁺=1000

18.2-{(1E,3E,5E,7E)-7-[1-(5-Carboxypentyl)-4,5,6,7-tetrafluoro-3-methyl-3-(4-sulfobutyl)-1,3-dihydro-2H-indol-2-ylidene]hepta-1,3,5-trienyl}-3-methyl-1,3-bis(4-sulfobutyl)-4,6-bis(trifluoromethyl)-3H-indolium(Compound 18)

18.1 4,6-Bis(trifluoromethyl)-2,3-dimethyl-3-(4-sulfobutyl)-3H-indole

To 3,5-bis(trifluoromethyl)phenyl hydrazine (1 g) in acetic acid (30 ml)was added 5-methyl-6-oxoheptane-1-sulphonic acid (1.5 g) and thesolution heated to reflux for 3 days. The volatiles were removed on arotary evaporator to give the crude product, 0.8 g of which was purifiedby preparative HPLC. The relevant fractions were combined, concentratedon a rotary evaporator and freeze dried to give the desired product (60mg). M⁺=417

18.24,6-Bis(trifluoromethyl)-2,3-dimethyl-1,3-bis(4-sulfobutyl)-3H-indolium

To 4,6-bis(trifluoromethyl)-2,3-dimethyl-3-(4-sulfobutyl)-3H-indole (27mg) was added butane sultone (2 ml) and the solution heated to 140° C.for 3 days. On cooling, the product was extracted into water andpurified by preparative HPLC. The relevant fractions were combined,concentrated on a rotary evaporator and freeze dried to give the desiredproduct (7 mg). M⁺=554

18.36-[(2E)-4,5,6,7-tetrafluoro-3-methyl-2-[(2E,4E,6E)-6-(phenylimino)hexa-2,4-dienylidene]-3-(4-sulfobutyl)-2,3-dihydro-1H-indol-1-yl]hexanoicacid

To1-(5-carboxypentyl)-4,5,6,7-tetrafluoro-3-methyl-3-(4-sulfobutyl)-3H-indolium(24 mg) was added N-[5-(phenylamino)-2,4-pentadienylidenemonohydrochloride (13 mg), acetic anhydride (4 ml) and acetic acid (2ml). This mixture was heated to 120° C. for 1 hour 45 minutes afterwhich time it was cooled to room temperature. This crude reactionmixture (ca. 5 mg/ml product) was used without purification as furtherdescribed.

18.42-{(1E,3E,5E,7E)-7-[1-(5-Carboxypentyl)-4,5,6,7-tetrafluoro-3-methyl-3-(4-sulfobutyl)-1,3-dihydro-2H-indol-2-ylidene]hepta-1,3,5-trienyl}-3-methyl-1,3-bis(4-sulfobutyl)-4,6-bis(trifluoromethyl)-3H-indolium(Compound 18)

To6-[(2E)-4,5,6,7-tetrafluoro-3-methyl-2-[(2E,4E,6E)-6-(phenylimino)hexa-2,4-dienylidene]-3-(4-sulfobutyl)-2,3-dihydro-1H-indol-1-yl]hexanoicacid (1.6 ml of ca. 5 mg/ml) was added4,6-bis(trifluoromethyl)-2,3-dimethyl-1,3-bis(4-sulfobutyl)-3H-indolium(7 mg) and potassium acetate (10 mg). On agitation overnight,preparative HPLC was performed, the relevant fractions were combined,concentrated on a rotary evaporator and freeze dried to give the desiredproduct (1 mg). M⁺=1083

19.2-{(1E,3E,5E,7E)-7-[1-(5-Carboxypentyl)-4,5,6,7-tetrafluoro-3-methyl-3-(4-sulfobutyl)-1,3-dihydro-2H-indol-2-ylidene]hepta-1,3,5-trienyl}-3-methyl-1,3-bis(4-sulfobutyl)-5-trifluoromethyl-3H-indolium(Compound 19)

19.1 5-Trifluoromethyl-2,3-dimethyl-3-(4-sulfobutyl)-3H-indole

To 4-(trifluoromethyl)phenyl hydrazine (2.18 g) in acetic acid (30 ml)was added 5-methyl-6-oxoheptane-1-sulphonic acid (4 g) and the solutionheated to reflux overnight. The volatiles were removed on a rotaryevaporator to give the crude product, 0.44 g of which was purified bypreparative HPLC. The relevant fractions were combined, concentrated ona rotary evaporator and freeze dried to give the desired product (180mg). M⁺=349

19.2 5-Trifluoromethyl-2,3-dimethyl-1,3-bis(4-sulfobutyl)-3H-indolium

To 5-trifluoromethyl-2,3-dimethyl-3-(4-sulfobutyl)-3H-indole (50 mg) wasadded butane sultone (2 ml) and the solution heated to 140° C. for 3days. On cooling, the product was extracted into water and purified bypreparative HPLC. The relevant fractions were combined, concentrated ona rotary evaporator and freeze dried to give the desired product (29mg). M⁺=486

19.32-{(1E,3E,5E,7E)-7-[1-(5-Carboxypentyl)-4,5,6,7-tetrafluoro-3-methyl-3-(4-sulfobutyl)-1,3-dihydro-2H-indol-2-ylidene]hepta-1,3,5-trienyl}-3-methyl-1,3-bis(4-sulfobutyl)-5-trifluoromethyl-3H-indolium(Compound 19)

To6-[(2E)-4,5,6,7-tetrfluoro-3-methyl-2-[(2E,4E,6E)-6-(phenylimino)hexa-2,4-dienylidene]-3-(4-sulfobutyl)-2,3-dihydro-1H-indol-1-yl]hexanoicacid (1.6 ml of ca. 5 mg/ml) was added5-Trifluoromethyl-2,3-dimethyl-1,3-bis(4-sulfobutyl)-3H-indolium (6 mg)and potassium acetate (10 mg). On agitation overnight, preparative HPLCwas performed, the relevant fractions were combined, concentrated on arotary evaporator and freeze dried to give the desired product (2.8 mg).M⁺=1015

20.2-{(1E,3E)-3-[1-(5-Carboxypentyl)-4,5,6,7-tetrafluoro-3-methyl-3-(4-sulfobutyl)-1,3-dihydro-2H-indol-2-ylidene]prop-1-enyl}-4,5,6,7-tetrafluoro-3-methyl-1,3-bis(4-sulfobutyl)-3H-indolium(Compound 20)

20.1 To1-(5-carboxypentyl)-2,3-dimethyl-3-(4-sulfobutyl)-4,5,6,7-tetrafluoro-3H-indolium(2 mg) was added2,3-dimethyl-4,5,6,7-tetrafluoro-1,3-bis(4-sulphonatobutyl)-3H-indolium(2.3 mg), ethanol (0.5 ml), triethyl orthoformate (100 ul) and pyridine(20 ul). The mixture was heated on an oil bath at 120° C. for 4 hours,after which time it was left at room temperature overnight. The mixturewas diluted with water and preparative HPLC was performed. The relevantfractions were combined, concentrated on a rotary evaporator and freezedried to give the desired product (0.1 mg). M⁺=967. A further compoundwas also isolated as described in 21.

21.1-(5-Carboxypentyl)-2-{(1E,3E)-3-[1-(5-carboxypentyl)-4,5,6,7-tetrafluoro-3-methyl-3-(4-sulfobutyl)-1,3-dihydro-2H-indol-2-ylidene]prop-1-enyl}-4,5,6,7-tetrafluoro-3-methyl-3-(4-sulfobutyl)-3H-indolium(Compound 21)

21.1 The experiment described in 20.1 also gave this material isolatedvia preparative HPLC (0.1 mg). M⁺=945

22.3-(5-Carboxypentyl)-3-methyl-2-{(1E,3E,5E)-5-[3-methyl-1,3-bis(4-sulfobutyl)-1,3-dihydro-2H-indol-2-ylidene]penta-1,3-dienyl}-5-sulfo-1-(4-sulfobutyl)-3H-indolium(Compound 22)

22.1 2,3-Dimethyl-1,3-bis(4-sulphonatobutyl)-3H-indolium, disodium salt

Sulphonated indole (˜5 g, 18 mmol) and butane sultone (12.2 g, 90 mmol,5 eq.) were stirred at 140° for 4 hours. The reaction mixture wasallowed to cool and then poured into stirring ethyl acetate. Thematerial did not precipitate, but remained as an oil from which theethyl acetate was decanted.

Yield: ˜10 g. M⁺=418.

22.23-(5-Carboxypentyl)-3-methyl-2-{(1E,3E,5E)-5-[3-methyl-1,3-bis(4-sulfobutyl)-1,3-dihydro-2H-indol-2-ylidene]penta-1,3-dienyl}-5-sulfo-1-(4-sulfobutyl)-3H-indolium(Compound 22)

2,3-Dimethyl-3-(5-carboxypentyl)-5-sulfo-1-(3-sulfobutyl)indolium,disodium salt (3.4 g, 6.84 mmol) was dissolved in acetic acid (25 ml)containing triethylamine (2.5 ml). Malonaldehyde bisphenylimine HCl(4.15 g, 16 mmol) was added and the mixture heated at 140° C. withstirring overnight. The reaction mixture was rotary evaporated to yielda red oil which was dissolved in water (50 ml) and acetonitrile (30 ml).This was filtered and purified to yield 1 g of dye intermediate. The dyeintermediate was dissolved in acetic acid:acetic acid (1:1, 50 ml)together with 1 equivalent of2,3-dimethyl-1,3-bis(4-sulphonatobutyl)-3H-indolium (disodium salt) andpotassium acetate (2 g). The reaction stirred at 120° for 4 hours, thenrotary evaporated to dryness and purified by reverse phase HPLC. Yield:85 mg. M⁺=929.

23. Photostability Studies

Photostability studies were performed as detailed below. Allfluorophores were dissolved in water at equimolar concentrations. Eachsolution was divided into further vials. Some vials were maintained in adark environment as control samples during the course of theexperiments. The others were exposed to a strong light source.

A Wallac light box (1295-013) was employed as the strong light source.Samples were maintained at 22 cm above the light source, with continuousexposure to light. The UV/visible spectrum of each sample was measuredonce every twenty four hours. The same cuvettes and spectrophotometerwere used for each measurement point. For the control samples maintainedin the dark, the UV/visible absorption spectra were measured at both thestart and at the end of the experiment. Seven sets of experiments wereperformed.

23.1 The photostabilities of Cy5F (Compound 1) and Compound 4 werestudied in comparison with a non-fluorinated analogue, Cy5.23.2 The photostability of Cy7F (Compound 2), Compound 18 and Compound19 was compared with a non-fluorinated analogue, Cy7.23.3 The photostabilities of Compound 3 and Compound 22 were comparedwith Alexa 647 (Invitrogen).23.4 The photostabilities of Compounds 5, 7, 9, 11 and 13 were comparedwith Alexa 647 (Invitrogen) and Cy5.23.5 The photostabilities of Compounds 6, 8, 10, 12 and 14 were comparedwith Cy5.23.6 The photostabilities of Compounds 15, 16 and 17 were compared withAlexa 647 (Invitrogen) and Cy5.23.7 The photostabilities of Compound 20 and Compound 21 were comparedwith Cy3.

The data for each experiment was normalised and plotted as shown inFIGS. 3, 4, 5, 6, 7, 8 and 9. The results demonstrate that thefluorinated dyes generally demonstrate greater resistance tophotobleaching when compared with the non-fluorinated dye analogues.Control reactions that were not exposed to light over the same period,showed no reduction in absorption.

The above examples illustrate specific aspects of the present inventionand are not intended to limit the scope thereof in any respect andshould not be so construed. Those skilled in the art having the benefitof the teachings of the present invention as set forth above, can effectnumerous modifications thereto. These modifications are to be construedas being encompassed within the scope of the present invention as setforth in the appended claims.

1. A compound of formula (I):

wherein: groups R¹, R², R¹¹, R¹², R¹³ and R¹⁴ are selected independentlyfrom -L-M, -L-P, C₁-C₆ alkyl and —(CH₂)_(k)—SO₃H, where k is an integerfrom 1 to 10; groups R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹ and R¹⁰ are selectedindependently from hydrogen, -L-M, -L-P, —SO₃H, —SO₂—CF₃ and—(CF₂)_(m)—F, where m is 0 or an integer from 1 to 4; or R³ taken incombination with R⁴ or R⁵ taken in combination with R⁶ and/or R⁷ takenin combination with R⁸ or R⁹ taken in combination with R¹⁰ form a fusedaromatic six-membered ring containing carbon atoms and being optionallysubstituted one or more times by —SO₃H, —SO₂—CF₃ or —(CF₂)_(m)—F, wherem is hereinbefore defined; L is a linking group having a chain from 1-20linked atoms selected from the group consisting of carbon, nitrogen,oxygen and sulphur atoms; M is a target bonding group; P is a conjugatedcomponent; groups R¹⁵ are hydrogen or two or more of R¹⁵ groups arecombined to form a one-ring or two-fused hydrocarbon ring system eachring having five or six carbon atoms, and remaining groups R¹⁵ arehydrogen; and n is an integer from 1 to 3; provided that: i) at leastone of groups R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰ R¹¹, R¹², R¹³ andR¹⁴ is -L-M or -L-P; ii) at least one of groups R³, R⁴, R⁵, R⁶, R⁷, R⁸,R⁹ and R¹⁰ comprises fluorine; and iii) when any one of R³, R⁴, R⁵, R⁶,R⁷, R⁸, R⁹ and R¹⁰ is —SO₃H at least one of groups R¹¹, R¹², R¹³ and R¹⁴is selected from -L-M, -L-P and —(CH₂)_(k)—SO₃H.
 2. The compound ofclaim 1, wherein: the groups R¹ and R² are selected independently from-L-M, -L-P, C₁-C₆ alkyl and —(CH₂)_(k)—SO₃H; groups R³, R⁴, R⁵, R⁶, R⁷,R⁸, R⁹ and R¹⁰ are selected independently from hydrogen, —SO₃H, —SO₂—CF₃and —(CF₂)_(m)—F, where m is 0 or an integer from 1 to 4; or R³ taken incombination with R⁴ or R⁵ taken in combination with R⁶ and/or R⁷ takenin combination with R⁸ or R⁹ taken in combination with R¹⁰ form a fusedaromatic six-membered ring containing carbon atoms and being optionallysubstituted one or more times by —SO₃H, —SO₂—CF₃ or —(CF₂)_(m)—F, wherem is hereinbefore defined; groups R¹¹, R¹²R¹³ and R¹⁴ are selectedindependently from C₁-C₆ alkyl and —(CH₂)_(k)—SO₃H; and groups R¹⁵, L, Mand P, k and n are defined as in claim 1; provided that: i) at least oneof groups R¹ and R² is -L-M or -L-P; ii) at least one of groups R³, R⁴,R⁵, R⁶, R⁷, R⁸, R⁹ and R¹⁰ comprises fluorine; and iii) when any one ofR³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹ and R¹⁰ is —SO₃H at least one of groups R¹¹,R¹², R¹³ and R¹⁴ is —(CH₂)_(k)—SO₃H.
 3. The compound of claim 1,wherein: groups R¹¹, R¹², R¹³ and R¹⁴ are selected independently from-L-M, -L-P, C₁-C₆ alkyl and —(CH₂)_(k)—SO₃H, groups R³, R⁴, R⁵, R⁶, R⁷,R⁸, R⁹ and R¹⁰ are selected independently from hydrogen, —SO₃H, —SO₂—CF₃and —(CF₂)_(m)—F, where m is 0 or an integer from 1 to 4; or R³ taken incombination with R⁴ or R⁵ taken in combination with R⁶ and/or R⁷ takenin combination with R⁸ or R⁹ taken in combination with R¹⁰ form a fusedaromatic six-membered ring containing carbon atoms and being optionallysubstituted one or more times by —SO₃H, —SO₂—CF₃ or —(CF₂)_(m)—F, wherem is hereinbefore defined; and groups R¹ and R² are selectedindependently from C₁-C₆ alkyl and —(CH₂)_(k)—SO₃H; provided that: i) atleast one of groups R¹¹, R¹², R¹³ and R¹⁴ is -L-M or -L-P; and ii) atleast one of groups R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹ and R¹⁰ comprisesfluorine; and iii) when any one of R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹ and R¹⁰ is—SO₃H at least one of groups R¹¹, R¹², R¹³ and R¹⁴ is —(CH₂)_(k)—SO₃H.4. The compound of claim 1, wherein at least one of groups R³, R⁴, R⁵,R⁶, R⁷, R⁸, R⁹ and R¹⁰ is fluorine.
 5. The compound of claim 1, whereintwo or more groups R¹⁵ form a hydrocarbon ring system of formula:

wherein n is an integer from 1 to 3 and p is 0 or 1; and remaininggroups R¹⁵ are hydrogen.
 6. The compound of claim 1, wherein each groupR¹⁵ is hydrogen.
 7. The compound of claim 1, wherein L is a linkinggroup having the formula:—(CHR′)_(p)-Q-(CHR′)_(r)— where Q is selected from: —CHR′—, —NR′—, —O—,—S—, CR′═CR′—, —Ar—, —C(O)—NR′— and —C(O)—O—, wherein R′ is hydrogen orC₁-C₄ alkyl; Ar is phenylene, optionally substituted with sulphonate; pis 0-5 and r is 1-5.
 8. The compound of claim 7, wherein Q is selectedfrom: —CHR′— and —C(O)—NH—.
 9. The compound of claim 7, wherein Q is—CHR′— and R′ is hydrogen.
 10. The compound of claim 1, wherein -L-Mand/or -L-P comprise a carboxypentyl group. 11-54. (canceled)
 55. Acompound selected from: i)2-{(1E,3E,5E)-5-[1-(5-Carboxypentyl)-4,5,6,7-tetrafluoro-3-methyl-3-(4-sulfobutyl)-1,3-dihydro-2H-indol-2-ylidene]penta-1,3-dienyl}-4,5,6,7-tetrafluoro-3-methyl-1,3-bis(4-sulfobutyl)-3H-indolium(Cy5F) (Compound 1); ii)2-{(1E,3E,5E,7E)-7-[1-(5-Carboxypentyl)-4,5,6,7-tetrafluoro-3-methyl-3-(4-sulfobutyl)-1,3-dihydro-2H-indol-2-ylidene]hepta-1,3-5-trienyl}-4,5,6,7-tetrafluoro-3-methyl-1,3-bis(4-sulfobutyl)-3H-indolium(Cy7F) (Compound 2); iii)3-(5-Carboxypentyl)-3-methyl-5-sulfo-1-(4-sulfobutyl)-2-{1E,3E,5E}-5-[4,5,6,7-tetrafluoro-3-methyl-1,3-bis(4-sulfobutyl)-1,3-dihydro-2H-indol-2-ylidene]penta-1,3-dienyl}-3H-indolium(Compound 3); iv)1-(5-Carboxypentyl)-3-methyl-5-sulfo-3-(4-sulfobutyl)-2-{(1E,3E,5E)-5-[4,5,6,7-tetrafluoro-3-methyl-1,3-bis(4-sulfobutyl)-1,3-dihydro-2H-indol-2-ylidene]penta-1,3-dienyl}-3H-indolium(Compound 4); v)2-{(1E,3E,5E)-5-[1-(5-Carboxypentyl)-4,5,6,7-tetrafluoro-3-methyl-3-(4-sulfobutyl)-1,3-dihydro-2H-indol-2-ylidene]penta-1,3-dienyl}-4,6-difluoro-3-methyl-1,3-bis(4-sulfobutyl)-3H-indolium(Compound 5); vi)2-{(1E,3E,5E)-5-[1-(5-Carboxypentyl)-4,6-difluoro-3,3-dimethyl-1,3-dihydro-2H-indol-2-ylidene]penta-1,3-dienyl}-4,5,6,7-tetrafluoro-3-methyl-1,3-bis(4-sulfobutyl)-3H-indolium(Compound 6); vii)1-(5-Carboxypentyl)-2-{(1E,3E,5E)-5-[1-(5-carboxypentyl)-4,6-difluoro-3,3-dimethyl-1,3-dihydro-2H-indol-2-ylidene]penta-1,3-dienyl}-4,5,6,7-tetrafluoro-3-methyl-3-(4-sulfobutyl)-3H-indolium(Compound 7); viii)2-{(1E,3E,5E)-5-[1-(5-Carboxypentyl)-4,5,6,7-tetrafluoro-3-methyl-3-(4-sulfobutyl)-1,3-dihydro-2H-indol-2-ylidene]penta-1,3-dienyl}-3-methyl-1,3-bis(4-sulfobutyl)-5-[(trifluoromethyl)sulfonyl]-3H-indolium(Compound 8); ix)2-{(1E,3E,5E)-5-[1-(4-Sulfobutyl)-4,6-bis(trifluoromethyl)-3-methyl-3-(5-carboxypentyl))-1,3-dihydro-2H-indol-2-ylidene]penta-1,3-dienyl}-4,6-difluoro-3-methyl-1,3-bis(4-sulfobutyl)-3H-indolium(Compound 9); x)2-{(1E,3E,5E)-5-[1-(5-Carboxypentyl)-4,5,6,7-tetrafluoro-3-methyl-3-(4-sulfobutyl)-1,3-dihydro-2H-indol-2-ylidene]penta-1,3-dienyl}-3-methyl-1,3-bis(4-sulfobutyl)-6-(trifluoromethyl)-3H-indolium(Compound 10); xi)2-{(1E,3E,5E)-5-[1-(5-Carboxypentyl)-4,5,6,7-tetrafluoro-3-methyl-3-(4-sulfobutyl)-1,3-dihydro-2H-indol-2-ylidene]penta-1,3-dienyl}-4,5,6,7,8,9-hexafluoro-1,1-dimethyl-3-(4-sulfobutyl)-1H-benzo[e]indolium(Compound 11); xii)2-{(1E,3E,5E)-5-[1-(5-Carboxypentyl)-4,6-difluoro-3,3-dimethyl-1,3-dihydro-2H-indol-2-ylidene]penta-1,3-dienyl}-6-fluoro-3-methyl-1,3-bis(4-sulfobutyl)-4-(trifluoromethyl)-3H-indolium(Compound 12); xiii)2-{(1E,3E,5E)-5-[1-(5-Carboxypentyl)-4,6-difluoro-3,3-dimethyl-1,3-dihydro-2H-indol-2-ylidene]penta-1,3-dienyl}-4,6-difluoro-3-methyl-1,3-bis(4-sulfobutyl)-3H-indolium(Compound 13); xiv)2-{(1E,3E,5E)-5-[1-(5-Carboxypentyl)-4,6-difluoro-3,3-dimethyl-1,3-dihydro-2H-indol-2-ylidene]penta-1,3-dienyl}-3-methyl-5-sulfo-1,3-bis(4-sulfobutyl)-3H-indolium(Compound 14); xv)2-{(1E,3E,5E)-5-[3-(5-Carboxypentyl)-4,5,6,7-tetrafluoro-3-methyl-1-(4-sulfobutyl)-1,3-dihydro-2H-indol-2-ylidene]penta-1,3-dienyl}-4,5,6,7-tetrafluoro-3-methyl-1,3-bis-(4-sulfobutyl)-3H-indolium(Compound 15); xvi)2-{(1E,3E,5E)-5-[3-(5-Carboxypentyl)-6-fluoro-3-methyl-1-(4-sulfobutyl)-4-(trifluoromethyl)-1,3-dihydro-2H-indol-2-ylidene]penta-1,3-dienyl}-4,5,6,7-tetrafluoro-3-methyl-1,3-bis(4-sulfobutyl)-3H-indolium(Compound 16); xvii)2-{(1E,3E,5E)-5-[3-(5-Carboxypentyl)-4-trifluoromethyl-6-fluoro-3-methyl-1,3-dihydro-2H-indol-2-ylidene]penta-1,3-dienyl}-3-(5-carboxypentyl)-4-trifluoromethyl-6-fluoro-3-methyl-3H-indolium(Compound 17); xviii)2-{(1E,3E,5E,7E)-7-[1-(5-Carboxypentyl)-4,5,6,7-tetrafluoro-3-methyl-3-(4-sulfobutyl)-1,3-dihydro-2H-indol-2-ylidene]hepta-1,3,5-trienyl}-3-methyl-1,3-bis(4-sulfobutyl)-4,6-bis(trifluoromethyl)-3H-indolium(Compound 18); xix)2-{(1E,3E,5E,7E)-7-[1-(5-Carboxypentyl)-4,5,6,7-tetrafluoro-3-methyl-3-(4-sulfobutyl)-1,3-dihydro-2H-indol-2-ylidene]hepta-1,3,5-trienyl}-3-methyl-1,3-bis(4-sulfobutyl)-5-trifluoromethyl-3H-indolium(Compound 19); xx)2-{(1E,3E)-3-[1-(5-Carboxypentyl)-4,5,6,7-tetrafluoro-3-methyl-3-(4-sulfobutyl)-1,3-dihydro-2H-indol-2-ylidene]prop-1-enyl}-4,5,6,7-tetrafluoro-3-methyl-1,3-bis(4-sulfobutyl)-3H-indolium(Compound 20); and xxi)1-(5-Carboxypentyl)-2-{(1E,3E)-3-[1-(5-carboxypentyl)-4,5,6,7-tetrafluoro-3-methyl-3-(4-sulfobutyl)-1,3-dihydro-2H-indol-2-ylidene]prop-1-enyl}-4,5,6,7-tetrafluoro-3-methyl-3-(4-sulfobutyl)-3H-indolium(Compound 21). 56-57. (canceled)
 58. The compound of claim 1, wherein—(CH₂)_(k)—SO₃H is selected from —(CH₂)₃—SO₃H and —(CH₂)₄—SO₃H.
 59. Thecompound of claim 1, wherein at least two of groups R³, R⁴, R⁵ and R⁶and/or groups R⁷, R⁸, R⁹ and R¹⁰ are F and any remaining groups R³, R⁴,R⁵, R⁶, R⁷, R⁸, R⁹ or R¹⁰ are H.
 60. The compound of claim 1, whereingroups R³, R⁴, R⁵ and R⁶ and/or groups R⁷, R⁸, R⁹ and R¹⁰ are F.
 61. Thecompound of claim 1, wherein at least one of groups R³, R⁴, R⁵ and R⁶and/or groups R⁷, R⁸, R⁹ and R¹⁰ are perfluoro C₁-C₄ alkyl and anyremaining groups R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹ and R¹⁰ are selected from Hor F.
 62. The compound of claim 61, wherein not more than two of the R³,R⁴, R⁵ and R⁶ positions and/or the R⁷, R⁸, R⁹ and R¹⁰ positions aresubstituted by perfluoro C₁-C₄ alkyl.
 63. The compound of claim 61,wherein said perfluoro C₁-C₄ alkyl is trifluoromethyl.
 64. The compoundof claim 1, wherein at least one of groups R³, R⁴, R⁵ and R⁶ and/orgroups R⁷, R⁸, R⁹ and R¹⁰ is —SO₂—CF₃ and any remaining groups R³, R⁴,R⁵, R⁶, R⁷, R⁸, R⁹ and R¹⁰ are selected from H or F.
 65. The compound ofclaim 1, wherein said target bonding group M comprises a reactive groupfor reaction with a functional group on a target material, or afunctional group for reaction with a reactive group on a targetmaterial.
 66. The compound of claim 65, wherein said reactive group isselected from the group consisting of succinimidyl ester,sulpho-succinimidyl ester, 4-sulfo-2,3,5,6-tetrafluorophenol (STP)ester, isothiocyanate, maleimide, haloacetamide, acid halide, hydrazide,vinylsulphone, dichlorotriazine and phosphoramidite.
 67. The compound ofclaim 65, wherein said functional group is selected from the groupconsisting of hydroxy, amino, sulphydryl, imidazole, carbonyl includingaldehyde and ketone, carboxylic acid and thiophosphate.
 68. The compoundof claim 1, wherein said target bonding group M comprises an affinitytag.
 69. The compound of claim 1, wherein P is selected from the groupconsisting of antibody, lipid, protein, peptide, carbohydrate,nucleotides which contain or are derivatized to contain one or more ofan amino, sulphydryl, carbonyl, hydroxyl, carboxylic acid andthiophosphate groups, and oxy or deoxy polynucleic acids which containor are derivatized to contain one or more of an amino, sulphydryl,carbonyl, hydroxyl, carboxylic acid and thiophosphate groups, microbialmaterials, drugs, hormones, cells, cell membranes and toxins.